Methods and compositions for DNA manipulation

ABSTRACT

single-stranded extension having a desired length and sequence composition. Methods for forming single-stranded extensions include: the use of a cassette containing at least one nicking site and at least one restriction site at a predetermined distance from each other and in a predetermined orientation; or primer-dependent amplification which introduces into a polynucleotide molecule, a modified nucleotide which is excised to create a nick using a nicking agent. The methods and compositions provided can be used to manipulate a DNA sequence including introducing site specific mutations into a polynucleotide molecule and for cloning any polynucleotide molecule or set of joined polynucleotide molecules in a recipient molecule such as a vector of choice.

CROSS REFERENCE

This Application is a continuation of U.S. application Ser. No.12/192,503 filed Aug. 15, 2008 (now abandoned), which is a divisionalapplication of U.S. application Ser. No. 10/407,637 filed Apr. 4, 2003,now U.S. Pat. No. 7,435,572 issued Oct. 14, 2008, which claims priorityfrom U.S. Provisional Application Ser. No. 60/372,352 filed Apr. 12,2002, U.S. Provisional Application Ser. No. 60/372,675 filed Apr. 15,2002 and U.S. Provisional Application Ser. No. 60/421,010 filed Oct. 24,2002, all of which are herein incorporated by reference.

TECHNICAL FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to compositions and methods for cloningand/or manipulating target DNA molecules at a desired sequence in asingle experimental format.

In the prior art, methods for manipulation and cloning of DNA include:amplification of DNA by Polymerase Chain Reaction (PCR); cleavage of DNAwith restriction endonucleases; and ligation to create recombinantmolecules. Limitations of these techniques include: lack of suitablerestriction sites in DNA which creates experimental difficulties inaccessing a desired location of the DNA sequence for a particularmanipulation; poor yield of vector compatible molecules which arisesfrom the template-independent terminal transferase activity ofpolymerase which introduces a non-template nucleotide at the 3′-terminiof amplified products (Clark, Nucl. Acid. Res., 16:9677-9686 (1988));and incompatibility of the termini of the amplified fragments withtermini of recipient molecules thereby preventing efficient insertion ofthe fragments into vectors or fusion of two or more PCR products to oneanother. Addition of the 3′ nucleotide as a result of terminaltransferase activity of the polymerase may be overcome by end polishingof PCR products using a polymerase with 3′ to 5′ exonuclease activity(Hemsley, et al., Nucl. Acid. Res., 17:6545-6551 (1989)). Alternatively,specially prepared vectors that carry 3′-T overhangs may be used toclone PCR products carrying non-template adenine at the 3′ ends(Marchuk, et al. Nucl. Acid. Res., 19:1154 (1990)). However, both theblunt-end insertion and the T/A overhang insertion are inefficient andthe above methods do not permit control over the orientation of theinserted fragment in the vector.

Efforts to improve the efficiency of blunt-end insertion of a DNA ofinterest by eliminating the background of self-ligated vector, includeperforming an insertion into a SmaI-linearized vector in the presence ofSmaI restriction endonuclease which will cleave the self-ligated vectormolecules at the re-generated SmaI sites (Liu, et al., BioTechniques,12:28-30 (1992)). The PCR-Script Cloning Systems of Stratagene, Ltd. (LaJolla, Calif.) uses the rare-cleavage restriction enzyme SrfI for asimilar purpose. The above methods are ineffective if the PCR productincludes any internal SmaI or SrfI sites respectively, or if the site isre-generated after the insertion into the vector DNA.

Another cloning methodology involves preparing amplified segments of DNAfor which different restriction sites are added to the 5′-ends of theamplification primers so as to incorporate these sites into the PCRproducts during amplification (Scharf, et al. Science, 233:1076-1078(1986)). The cleavage of PCR product and vector DNA by the samerestriction endonuclease produces compatible single-stranded terminithat can be joined by DNA ligase. This method has severaldisadvantages 1) if the restriction site that is introduced into theprimer is present somewhere within the PCR product, the internal sitewill also be cleaved during endonuclease digestion, thus, preventingcloning of full-length PCR products; 2) many restriction endonucleasesinefficiently cleave sites close to the end of DNA fragment (Kaufman, etal. BioTechniques, 9:304-306 (1992)), therefore it is necessary to add3-6 additional nucleotides to the 5′-ends of primers to ensure efficientcleavage by a particular restriction endonuclease; 3) many restrictionendonucleases are inhibited by particular components in theamplification reaction, for example, some restriction endonucleases areinhibited by single-stranded PCR primers, so an additional PCR productpurification step is necessary before restriction endonucleasedigestion; and 4) often restriction endonuclease generated termini areself-complementary resulting in side-products during the ligationreaction thus greatly reducing the yield of target product.

To overcome these limitations, several restriction endonuclease-freetechniques have been described that allow creation of single-strandedtermini on the PCR products. U.S. application Ser. No. 09/738,444describes the use of nicking endonucleases to create single-strandedextensions that may be used specifically to join fragments withcomplementary ends.

Single-stranded termini complementary to the AccI and XmaI restrictionendonuclease termini were generated by using the 3′ to 5′ exonucleaseactivity of T4 DNA Polymerase (Stoker, Nucl. Acid. Res., 18:4290(1990)). In the presence of only dATP and dTTP, the exonuclease activityis limited to removal of only G and C nucleotides, thus creating therequisite single-stranded termini for sub-cloning into a AccI- andXmaI-cleaved plasmid vector. In technology referred to asLigation-Independent Cloning of PCR products (LIC-PCR) (Aslanidis, etal., Nucl. Acid. Res., 18:6069-6074 (1990)) target DNA is amplified withprimers containing 12 additional nucleotides at their 5′ ends that lackcytosine. As a result, the PCR product on the 3′ ends is flanked by a12-nucleotide sequence lacking guanine. Treatment of the PCR productwith the 3′ to 5′ exonuclease associated with T4 DNA Polymerase in thepresence of dGTP removes the 3′ terminal sequences until the first dGMPresidue is reached, thus leaving a 12 nucleotide 5′ single-strandedextension. However disadvantages of this technology include the need fora special vector having compatible 12 nucleotide 5′ single-strandedextensions for cloning. The preparation of such vectors includeamplification of the entire vector with primers containing 12 nucleotidetails complementary to the tails used for amplification of targetfragment and subsequently treating the amplified vector with T4 DNAPolymerase to create complementary 12 nucleotide long single-strandedextensions. A modified technique of LIC-PCR has been described, wherethe specific sequences devoid of particular bases are engineered intoplasmid vectors to replace the vector amplification step by arestriction digestion step (Haun, et al. BioTechniques, 13:515-518(1992); Kuijper, et al. Gene, 112:147-155 (1992); Cooney, BioTechniques,24:30-33 (1998)).

A disadvantage of the above-described methods is the need to removeleftover dNTP, before subjecting the PCR product to exonucleasetreatment. The use of this technology is limited to sequences devoid ofat least one nucleotide, and in addition the use of non-specificexonucleases to manipulate DNA may give rise to sequence rearrangementsat the position of vector-product junction of recovered recombinantmolecules.

Single-stranded overhangs or extensions have been produced during PCR byincorporating the non-base residue, 1,3-propanediol, into primersequences. This has the effect of terminating DNA synthesis (Kaluz, S.et al. (1994) Nucl. Acid. Res., 22, 4845). During PCR, Taq DNAPolymerase stops at the non-replicable element, leaving a portion of theprimer as a single strand. Since 1,3-propanediol also inhibits DNAreplication processes in vivo, the repair machinery of the bacterialhost has to remove the non-replicable element potentially causingunwanted sequence rearrangements in the recovered recombinant molecules.

Cloning and manipulating genes with the use of a DNA repair enzyme,Uracil DNA Glycosylase (UDG), has been described. (Rashtchian et al.U.S. Pat. No. 5,137,814; Berninger, U.S. Pat. No. 5,229,283; Nisson, etal., PCR Methods & Applications, 1:120-123 (1991); Rashtchian, et al.,PCR Methods & Applications, 2:124-130 (1992); Booth, et al., Gene,146:303-308 (1994); Rashtchian, Current Biology, 6:30-36 (1995)) UDGrecognizes uracil lesions in single- or double-stranded DNA and cleavesthe N-glycosylic bond between the deoxyribose moiety and the baseleaving an abasic site. During PCR, Taq DNA Polymerase insertsdeoxyadenisine opposite a deoxyuridine (U) lesion. Target DNA andcloning vectors can be amplified with primers at the 5′ ends carryingdUMP-containing tails. Subsequent treatment with the UDG glycosylaseresults in formation of multiple abasic sites on the ends of theamplified product. Strand separation across the modified portion of theamplified product and the vector that contains complementary ends(generated by the same approach) provides a re-annealed recombinantproduct having protruding single-stranded flaps which should be removedin vivo by the repair machinery of the bacterial host. Cloning of cDNAsby single-primer amplification (SPA) that employs a dU-containing primerhas been described in U.S. Pat. No. 5,334,515.

Since UDG does not cleave the phosphodiester backbone, the efficiency ofstrand separation to a great extent depends on the number of dUMPresidues within the 5′ ends of PCR products. Hence, at least one thirdof the 5′ tails of the PCR primer should consist of dUMP to achieveefficient strand separation between two strands of DNA duplex (U.S. Pat.No. 5,137,814 and U.S. Pat. No. 5,229,283). Another disadvantage of thismethod is that the entire plasmid vector must be amplified by PCR toproduce the linear vector flanked by the complementary extensionssuitable for sub-cloning of the UDG-treated PCR fragments.

UDG glycosylase has also been used to create Sad restrictionendonuclease-like cohesive ends on PCR fragments which are suitable forcloning into SacI-linearized vectors (Smith, et al. PCR Methods &Applications, 2:328-332 (1992); Watson, et al. BioTechniques, 23:858-862(1997)). However, this technology is very limited, as it allows cloningof PCR amplified product only into a Sad site. Another disadvantage ofthis method is that SacI-like cohesive termini are self-complementary.Therefore a variety of unwanted side-products are generated uponligation thus reducing the use of this technology in DNA manipulationsother than cloning of PCR products.

A chemical method for creating single-stranded overhangs on PCR productsemploys PCR primers containing ribonucleotides, such as rUMP or rCMP(Chen, et al. BioTechniques, 32:517-520 (2002); Jarell, et al. U.S. Pat.No. 6,358,712). After amplification, the PCR products are treated withrare-earth metal ions, such as La³⁺ or Lu³⁺ (Chen, et al. BioTechniques,32:517-520 (2002)) or sodium hydroxide (Jarell, et al. U.S. Pat. No.6,358,712) to hydrolyze the phosphodiester bond between thedeoxyribonucleotide and the ribonucleotide. Disadvantages include thehigh cost of PCR primers and in addition the vector DNA must be preparedby PCR with the use of primers containing ribonucleotides to generatecompatible termini suitable for sub-cloning.

Some of the PCR-based sub-cloning techniques described above can also beused for site-specific DNA mutagenesis. However their application islimited to cases in which, it is possible to introduce a specific changewithout disrupting the rest of the coding sequence. For example, whensuitable restriction sites are located in close proximity to thenucleotide sequence targeted for mutation, the PCR-basedoligonucleotide-directed site-specific mutagenesis is routinely used tointroduce desired mutations into target DNA sequences and the mutatedPCR fragment is then introduced in place of the wild-type sequence usingrestriction endonuclease digestion (Higuchi, et al. Nucl. Acid. Res.,16:7351-7367 (1988)). However, when the appropriate naturally occurringrestriction sites are not available, additional experimental proceduresmust be performed to introduce internal changes.

Another PCR-dependent mutagenesis method uses a “megaprimer”.Megaprimers are long, double-stranded DNAs which are often difficult todenature, to anneal and to extend to a full-length product.Consequently, the method has been found to be problematic when themegaprimer is longer than several hundred base pairs (Kammann, et al.Nucl. Acid. Res., 17:5404 (1989); Sarkar, et al. BioTechniques,8:404-407 (1990); Sarkar, et al. Nucl. Acid. Res., 20:4937-4938 (1992);Landt, et al. Gene, 96:125-128 (1990); Ling, et al. AnalyticalBiochemistry, 254:157-178 (1997); Smith, et al. BioTechniques,22:438-442 (1997); Colosimo, et al. BioTechniques, 26:870-873 (1999)).

Another PCR-dependent site-directed mutagenesis technique referred to as“overlap-extension” PCR has been described (Higuchi, et al. Nucl. Acid.Res., 16:7351-7367 (1988); Ho, et al. Gene, 77:51-59 (1989)). Twoprimary PCR reactions produce two overlapping DNA fragments, bothbearing the same mutation introduced via mutagenic primers in the regionof the overlap sequence. These fragments are then combined, denaturedand re-annealed to generate a hetero-duplex product via the overlappingsequence. In the re-annealed hetero-duplex product, the 3′ overlap ofeach strand serves as a primer for the extension of the complementarystrand. The extended full-size fusion is then amplified in a secondround of PCR using the outside primers. The overlap-extension method islaborious and inefficient in many practical applications for severalreasons: it requires the purification of intermediate PCR product toremove mutagenic primers; it requires two full rounds of PCR, whichincreases the possibility of introducing the undesired mutations; andthe efficiency of annealing heterologous molecules across the overlapregion is greatly reduced by the presence of a full-length complementarystrand of either fragment.

No existing single PCR-based method for site-directed mutagenesis andcloning appears to solve all of the problems associated with in vitroDNA manipulations. A single strategy for achieving a variety of DNAmanipulations, such as linking, adding, deleting or changing nucleotidesegments at any desired location of target DNA molecule would bedesirable.

SUMMARY OF THE INVENTION

In an embodiment of the invention, a method is provided for generating asingle-stranded extension on a polynucleotide molecule where thesingle-stranded extension has a desired length and sequence composition.The method includes the steps of: inserting a cassette into apolynucleotide molecule at a predetermined location; cleaving thepolynucleotide molecule with a nicking endonuclease specific for anicking site in the cassette and with a restriction endonucleasespecific for a restriction site in the cassette; and dissociating thecleaved polynucleotide molecule between the nicking site and therestriction site to generate the single-stranded extension with thedesired length and sequence composition.

In addition to the above described method, an additional embodiment ofthe invention is a cassette wherein the cassette includes adouble-stranded DNA having a nicking site located less than about 50nucleotides from a restriction site, the DNA being capable of insertioninto a polynucleotide molecule, wherein the restriction site in thecassette does not occur in the polynucleotide molecule.

In particular examples of the above embodiments, the method includes theuse of a cassette, where the cassette contains a nicking site and arestriction site for generating a 3′ or 5′, left side or right sidesingle-stranded extension; or one restriction site flanked on each sideby a nicking site for generating a left side and a right side 3′ or 5′single-stranded extension; or two restriction sites positioned betweentwo nicking sites for generating two single-stranded extensions. Thesequence between the nicking site and the restriction site determine thelength and composition of the single-stranded extension product. Thesingle-stranded extension produced is preferably no longer than about 20nucleotides. In those examples where two restriction sites are presentin a cassette, a spacer sequence may be located between the tworestriction sites. The spacer region may be selected according to itscoding sequence where it is desirable under certain circumstances thatthe spacer encode a marker and the polynucleotide molecule into whichthe cassette is inserted is a recipient molecule which is capable ofreplicating in a host cell. The spacer sequence provides a means todetermine in host cells transformed with a recipient molecule containingan inserted cassette, which recipient molecule contains a cassette andwhich recipient molecules lack a cassette or contain a defectivecassette.

In additional examples of the above embodiment, the cassette may containa nicking site positioned upstream (left side) or downstream (rightside) from the restriction site in the cassette in an orientationsuitable for nicking a first of two strands (top strand) or the secondof the two strands (bottom strand) with the nicking endonuclease. Incassettes where two nicking sites occur, these are each inverselyoriented with respect to each other.

In additional examples of the above embodiment, the recipient moleculemay be a vector and the vector may be selected from: pNEB205A, pNEB200A,pNEB210A, and pUC-TT.

In an additional embodiment of the invention, a nicking agent isprovided which includes a mixture of two or more enzymes wherein atleast one of the enzymes is a DNA glycosylase and at least one of theenzymes is a single-strand cleaving enzyme, wherein the nicking agent iscapable of excising a modified nucleotide from a polynucleotidemolecule.

In particular examples of the above embodiment, at least onesingle-stranded cleaving enzyme in the nicking agent generates a 5′phosphate in the polynucleotide molecule after excision of the modifiednucleotide. This activity is further exemplified by use of singlestranded cleaving enzymes: FPG glycosylase/AP lyase and Endo VIIIglycosylase/AP lyase. Alternatively or additionally, the nicking agentmay contain at least one single-stranded cleaving enzyme that generatesa 3′OH in the polynucleotide molecule after excision of the modifiednucleotide using, for example, EndoIV endonuclease.

The modified nucleotide described above may include deoxyuridine (U),8-oxo-guanine or deoxyinosine. Examples of nicking agents describedherein that are capable of excising these modified nucleotides include:for excising deoxyuridine(U)—UDG glycosylase in a mixture with EndoIVendonuclease; UDG glycosylase in a mixture with FPG glycosylase/APlyase; UDG glycosylase in a mixture with EndoVIII glycosylase/AP lyase;a mixture containing UDG glycosylase, EndoIV endonuclease and EndoVIIIglycosylase/AP lysase; for excising 8-oxo-guanine and deoxyuridine (U)—amixture containing UDG glycosylase, FPG glycosylase/AP lyase and EndoIVendonuclease; or UDG glycosylase in a mixture with FPG glycosylase/APlyase; and for excising deoxyinosine—AlkA glycosylase in a mixture withEndoVIII glycosylase/Ap lyase or AlkA glycosylase in a mixture with FPGglycosylase/AP lyase. In particular examples, the glycosylase and thesingle-strand cleaving enzyme are present in the nicking agent in anactivity ratio of at least about 2:1.

In an additional embodiment of the invention, a method is provided forgenerating a single-stranded extension on a polynucleotide molecule, thesingle-stranded extension having a desired length and composition. Themethod includes the steps of introducing into the polynucleotidemolecule at a specific location, a modified nucleotide; cleaving thepolynucleotide molecule at the modified nucleotide with a nicking agentto create a terminal sequence flanked by a nick; and dissociating theterminal sequence to generate the single-stranded extension with thedesired length and sequence composition.

In particular examples of the embodiment, the polynucleotide molecule isa product of primer pair dependent DNA amplification of a targetmolecule. Moreover, each primer in the primer pair may contain themodified nucleotide or alternatively one of the primers in the primerpair may contain the modified nucleotide. The composition of thesingle-stranded extension on the polynucleotide molecule may be suchthat it is complementary to a single-stranded extension on a secondpolynucleotide molecule.

In an additional embodiment of the invention, a method is provided forcreating a site-specific mutation in a target molecule. This embodimentincludes selecting two pairs of primers for amplifying the targetmolecule wherein one pair of primers produces one amplification productand the second pair of primers produces a second amplification product.One primer from each primer pair may contain a modified nucleotide andthe sequence of such primers complements each other at the 5′ end.Optionally one or both of these primers contain a mutation in thecomplementary or in a non-complementary 5′ sequence wherecomplementation is determined with respect to the target molecule. Thetarget molecule is then amplified using the two primer pairs to form twopolynucleotide molecules. These polynucleotide molecules are nicked atthe modified nucleotide with a nicking agent. The polynucleotidemolecules are then dissociated between the nick and the 5′ end toproduce 3′ single-stranded extensions on the two polynucleotidemolecules that are complementary to each other. The two polynucleotidemolecules are permitted to reassociate through the complementarysingle-stranded extensions to form a target molecule having a sitespecific mutation.

In an example of the above embodiment, the sequence at the 5′ end of theprimers adjacent to the modified nucleotide may be characterized asnon-complementary to the target molecule. In another example, one primerfrom each of the two primer pairs has a modified nucleotide positionedbetween a priming sequence and a 5′ terminal region, wherein the primingsequence is complementary to the target molecule and wherein the 5′terminal regions of the primers adjacent to the modified nucleotide arecomplementary to each other.

Additionally, the modified nucleotide on at least one primer may bepositioned at a junction between the priming sequence and the 5′terminal region. Alternatively, the modified nucleotide on at least oneprimer may be positioned between the 5′ sequence and an insertionsequence wherein the insertion sequence is adjacent to the primingsequence. In an additional configuration, the priming sequence on eachof the primers may complement sequences on the target molecule which areseparated by an intervening sequence.

The site-specific mutation referred to above may be an alteration in oneor more nucleotides or an inserted nucleotide sequence.

In an additional embodiment of the invention, an oligonucleotidesuitable for priming a DNA template is provided having a 5′ sequenceselected from GGAGACAU, GGGAAAGU, ACGAGACU, ACCAGACU and GGGGG(8-oxo-G)adjacent to a sequence identical to the 5′ end of the DNA template.

In an additional embodiment of the invention, a method is provided forjoining a plurality of linear polynucleotide molecules to form a singlemolecule. The method includes forming a single-stranded extension on oneor both ends of each of the plurality of polynucleotide molecule using acassette as described above. The product includes at least onesingle-stranded extension on one polynucleotide molecule that iscomplementary to a single-stranded extension on another polynucleotidemolecule. The plurality of polynucleotide molecules can then beassociated to form the single molecule.

In an additional embodiment, a method is provided for joining aplurality of linear polynucleotide molecules to form a single molecule.The method includes forming a single-stranded extension on one or bothends of a plurality of polynucleotide molecules using primer-dependentamplification described above. The product includes at least onesingle-stranded extension on one polynucleotide molecule that iscomplementary to a single-stranded extension on another polynucleotidemolecule. The plurality of polynucleotide molecules can then associateto form the single molecule.

In an additional embodiment of the invention, a method for inserting atarget molecule into a recipient molecule is provided. The methodincludes forming a first and a second-single-stranded extension on theends of the recipient molecule after cleavage of sites in a cassette.Single-stranded extensions are formed on a target molecule byprimer-dependent amplification. The single-stranded extension on one endof the target molecule is complementary to the first single-strandedextension on the recipient molecule; and on the other end of the targetmolecule is complementary to the second single-strand extension on therecipient molecule, so that the target molecule and the recipientmolecule associate to form a single target molecule.

In particular examples of the above embodiments, the target molecule maybe a product of joining a plurality of polynucleotide molecules, or mayrepresent individual conserved DNA domains such as Exons. In addition,the first- and the second single-stranded extensions on the recipientmolecule may have the same or different sequence.

In an embodiment of the present invention, a kit is provided thatincludes a nicking agent and a linearized vector. the kit may furtherinclude a DNA polymerase, a T4 DNA ligase or ast least one sequencingprimer.

In an embodiment of the present invention, a host cell is provided whichcontains a recipient molecule into which a target molecule has beeninserted via single-stranded extensions created according to the aboveembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows how a 3′ single-stranded extension of desired length andcomposition is generated on the left-side of a cleaved polynucleotidemolecule:

(a) inserted into a polynucleotide molecule is a cassette, havingboundaries (13), and containing site A recognized by nickingendonuclease A which nicks within site A on one strand as indicated bythe arrow and site B located downstream of site A recognized byrestriction endonuclease B, which cleaves both strands as indicated bythe arrows;

(b) the polynucleotide molecule containing the cassette showing breaksin the phosphodiester bond backbone from cleavage with nickingendonuclease A and restriction endonuclease B;

(c) the products of dissociation (1), (2) and (3) where (1) is theleft-side of the cleaved polynucleotide molecule flanked by a 3′single-stranded extension of the desired length and composition.

FIG. 2 shows how a 3′ single-stranded extension of desired length andcomposition can be generated on the right-side of a cleavedpolynucleotide molecule;

(a) inserted into a polynucleotide molecule is a cassette, havingboundaries (13), containing a site A which is inversely oriented to andlocated downstream of site B;

(b) the polynucleotide molecule containing the cassette showing breaksin the phosphodiester backbone from cleavage with nicking endonuclease Aand restriction endonuclease B;

(c) the products of dissociation (4), (5) and (6) where (4) is theright-side of the cleaved polynucleotide molecule flanked with a 3′single-stranded extension of a desired length and composition.

FIG. 3 shows how 3′ single-stranded extensions of desired length andcomposition can be obtained on both the left-side and the right-side ofa cleaved polynucleotide molecule:

(a) inserted into a polynucleotide molecule is a cassette, havingboundaries (13), containing two nicking sites A inversely oriented withrespect to each other and located on either side of a single restrictionendonuclease site B;

(b) the polynucleotide molecule containing the cassette showing breaksin the phosphodiester backbone from cleavage with nicking endonuclease Aand restriction endonuclease B;

(c) the products of dissociation (1), (3), (4) and (6) where (1) and (4)have 3′ single-stranded extensions of the desired length and nucleotidecomposition.

FIG. 4 shows how a 3′ single-stranded extension can be obtained on bothleft-side and right-side of a cleaved polynucleotide molecule:

(a) inserted into the polynucleotide molecule is a cassette, havingboundaries (13), and containing two inversely oriented nicking sites Aflanking two restriction sites B. Between the two restriction sites, isa spacer region of any desired length (shown by the dotted line);

(b) the polynucleotide molecule containing the cassette showing breaksin the phosphodiester backbone from cleavage with nicking endonuclease Aand restriction endonuclease B;

(c) the products of dissociation (1), (3), (4), (6) and (7) where (1)and (4) have 3′ single-stranded extensions of the desired length andnucleotide composition and (7) is the spacer region.

FIG. 5 shows how a 5′ single-stranded extension of desired length andcomposition can be generated on the left-side of a cleavedpolynucleotide molecule:

(a) inserted into the double-stranded polynucleotide molecule is acassette, having boundaries (13), containing a site C and a site D,where site C is recognized by a nicking endonuclease C which nicksoutside site C as indicated by an arrow. The site D located downstreamof site C is recognized by restriction endonuclease D, which cleavesboth strands as indicated by the arrows;

(b) the polynucleotide molecule containing the cassette showing breaksin the phosphodiester backbone from cleavage with nicking endonuclease Cand restriction endonuclease D;

(c) the products of dissociation (8), (9) and (10) where (8) is aleft-side of the cleaved polynucleotide molecule flanked by a 5′single-stranded extension of the desired length and composition.

FIG. 6 shows how 5′ single-stranded extensions can be obtained on bothleft-side and right-side of a cleaved polynucleotide molecule:

(a) inserted into the double-stranded polynucleotide molecule is acassette, having boundaries (13), and containing two nicking sites Cinversely oriented with respect to each other and located on either sideof a single restriction site D.

(b) the polynucleotide molecule containing the cassette showing breaksin the phosphodiester backbone from cleavage with nicking endonuclease Cand restriction endonuclease D;

(c) the products of dissociation (8), (10), (11) and (12) where (8) and(11) have 5′ single-stranded extensions of the desired length andnucleotide composition.

FIG. 7 shows how a target molecule can be inserted into a recipientmolecule in a predetermined orientation:

(a) a left and a right primer is shown where each primer at the 5′ endis characterized by: a plurality of nucleotides (N) in which Ncorresponds to any of A, T, G or C of a nucleotide sequence that is notcomplementary to a target molecule sequence, but is identical to thecorresponding left or right 3′ single-stranded extension on thelinearized recipient molecule except for the nucleotide “X”, which in aprimer sequence replaces the terminal 3′ nucleotide on the left andright end of the recipient molecule, and is the target for a nickingagent; and a plurality of nucleotides (n) that hybridize to thenucleotides on the complementary strand of the target molecule;

(b) the product of DNA amplification corresponding to the entiresequence of the left and right primers and the target molecule;

(c) the target molecule flanked by 3′ single-stranded extensionsproduced after treatment of amplified DNA with a nicking agent specificto X.

(d) the recipient molecule having 3′ single-stranded extensions whichare complementary to the 3′ single-stranded extensions on the targetmolecule of (c);

(e) the recombinant product resulting from combining the target andrecipient molecules of (c) and (d).

FIG. 8 shows how complementary 3′ single-stranded extensions can begenerated at the desired locations in a target molecule:

(a) design of primer pairs P2/P3 and P1/P4 showing their location in thetarget molecule, where Primers P1 and P2 overlap each other by apre-selected nucleotide sequence shown as a bold line, the pre-selectednucleotide sequence including a modified nucleotide X at the 3′ end ofthe overlap;

(b) the amplification products referred to as Fragments 1 and 2amplified using primer pairs P2/P3 and P1/P4 respectively and showingthe common overlap region (in black) with the presence of modifiednucleotide on the opposite strands in Fragments 1 and 2;

(c) the products of dissociation of Fragments 1 and 2 after cleavagewith a nicking agent specific to X having 3′ single-stranded extensionsthat are complementary to each other;

(d) Fragments 1 and 2 are re-associated through their complementary 3′single-stranded extensions.

FIGS. 9A and 9B shows how primer pairs (P2/P3 and P1/P4) can be modifiedto achieve a site-specific mutation in a target molecule. Only theprimer design step is shown. These primers can be used according to FIG.8 (b) to (d). X is the modified nucleotide:

FIG. 9A Primer design includes introducing a mutation (black triangle)in overlapping sequences of both P1 and P2 primers (shown in bold) suchthat when these primers are used in the generation of Fragments 1 and 2(FIG. 8, (b)) both amplified fragments will carry the mutation at thesame position of the common overlap region;

FIG. 9B Primer design includes introducing a mutation downstream of X inP1 only (black triangle). If P1 and P2 are used in the generation ofFragments 1 and 2 (FIG. 8, (b)) the mutation will be located withinFragment 2 downstream from the overlap region.

FIG. 10 shows how primer pairs (P2/P3 and P1/P4) can be modified toachieve a nucleotide sequence insertion in a target molecule. Only theprimer design step is shown. These primers can be used according to FIG.8 (b) to (d). The priming sequence (shown as arrows) in both overlappingPrimers P1 and P2 starts precisely at the position of the expectedinsertion. X is a modified nucleotide:

FIG. 10A Primer design includes Primers P1 and P2 having 5′ overlappingregions which correspond to an insertion sequence (shown in bold). X islocated at the junction between the priming sequence (shown as an arrow)and the insertion sequence.

FIG. 10B Primer design includes a Primer P2 having an entire insertionsequence at its 5′ end adjacent to the priming sequence (shown as adotted line plus a bold line). To create a common overlap, Primer P1contains the 5′ end of the insertion sequence (shown in bold). In P1, Xis located at the junction between the priming sequence (shown as anarrow) and the overlap region. In P2, X is located at the junction ofthe overlap region and the remainder of the insertion sequence.

FIG. 11 shows how primer pairs (P2/P3 and P1/P4) can be modified toachieve a nucleotide sequence deletion in a target molecule. Only theprimer design step is shown. These primers can be used according to FIG.8 (b) to (d).

Overlapping Primers P1 and P2 are designed so that priming sequence ofeach primer starts precisely beyond the targeted deletion region of thetarget molecule (dotted lines in the target molecule). To generate thecommon overlap region, an additional nucleotide sequence is included atthe 5′ end of Primer P2 which is the reverse-complement to the 5′ end ofP1. The overlapping regions are shown as a bold line. Each primercarries a single modified nucleotide X at the junction between theoverlapping region and the priming sequence.

FIG. 12 shows how primer pairs (P2/P3 and P1/P4) can be designed togenerate a fusion product from a target molecule A and a target moleculeB. Only the primer design step is shown. These primers can be usedaccording to FIG. 8 (b) to (d).

Overlapping Primers P1 and P2 are designed so that each primer annealsto a respective target molecule. To generate the common overlap region,an additional nucleotide sequence is included at the 5′ end of Primer P2which is the reverse-complement to the 5′ end of Primer P1. Theoverlapping regions are shown as a bold line. Each primer carries asingle modified nucleotide X at the junction between the overlappingregion and the priming sequence.

FIG. 13 shows how multiple primer pairs can be designed to achieveassembly of target molecule from the multiple intermediate targetmolecules following the approach in FIG. 12. For primer pairs P1/P2,P3/P4 and P5/P6 used to amplify intermediate target molecules A, B and Crespectively, two overlapping primer pairs, P2/P3, P4/P5 are designed asdescribed in FIG. 12.

FIG. 14 shows how multiple primer pairs can be designed to achieveassembly of a circular target molecule from multiple intermediate targetmolecules following the approach in FIG. 12. For primer pairs P1/P2,P3/P4, P5/P6, P7/P8 used to amplify target molecules A, B, C and Drespectively, four overlapping primer pairs, P2/P3, P4/P5, P6/P7 andP8/P1, are designed as described in FIG. 12.

FIG. 15 shows how to generate an eukaryotic gene directly from thegenomic DNA instead of making a cDNA by assembling individual Exonsusing multiple pairs of primers designed as shown in FIGS. 12 and 13.The 5′ common overlap region in the primer is shown in bold. The primingsequence is indicated by an arrow.

FIG. 16 shows how concurrent manipulation and cloning of target moleculecan be achieved:

(a) design of the outside Primers P3 and P4 permits insertion into arecipient molecule, while design of overlapping Primers P1 and P2permits manipulation of the target molecule. Outside Primers P3 and P4are designed as described in FIG. 7 (a). Overlapping Primers P1 and P2are designed as described in FIG. 9A. Target DNA is amplified as twooverlapping fragments with primer pairs P2/P3 and P1/P4;

(b) the amplification products identified as Fragment 1 and Fragment 2contain a newly introduced mutation within the common overlap region(black and white triangles);

(c) the dissociation products of Fragments 1 and 2 after cleavage with anicking agent specific for X, where Fragments 1 and 2 are flanked by 3′single-stranded extensions on both ends. The outside extensions arecomplementary to the 3′ single-stranded extensions on the recipientmolecule, while the inside extensions carrying a newly introducedmutation (white triangle) are complementary to each other;

(d) the recipient molecule is flanked by 3′ single-stranded extensionscomplementary to the outside 3′ single-stranded extensions of Fragments1 and 2;

(e) directional assembly of Fragment 1 and 2 into the recipient moleculeby means of annealing of complementary single-stranded extensions.

FIG. 17 shows how genomic DNA fragments outside the boundaries of knownsequences can be cloned into a recipient molecule:

(a) Primer 1 is specific for the target DNA close to the end of theknown sequence permitting linear amplification to producesingle-stranded polynucleotide molecules of different lengths;

(b) polyC tails are added to the 3′ ends of the amplifiedsingle-stranded polynucleotide molecules using terminal transferase anddCTP. PolyC tailed single-stranded fragments are amplified with Primer 2and Primer 3. Primer 2 has an 8-oxo-guanine at the junction between thepriming sequence and a 5′ polyG tail. Primer 3 is poly dG which iscomplementary to the polyC tail and contains an 8-oxo-guanine close tothe 5′ end at a position corresponding to the position of 8-oxo-guaninein Primer 2. “N”—A, G, C or T, whereas “H”—indicates A, T or C;

(c) the product of amplification with 8-oxo-guanine at the sixthposition from the 5′ end of the fragments;

(d) the amplified DNA is nicked by USER™ Enzyme 2 at 8-oxo-guanine thusgenerating 3′ single-stranded extensions of six cytosines on both endsof the amplified products;

(e) the amplified products representing the unknown genomic DNA flankedby 3′ single-stranded extensions of six cytosines are inserted into therecipient molecule which has 3′ single-stranded extensions consisting ofsix guanines.

FIG. 18 shows how a cDNA library can be created from mRNA by generatinga library of double-stranded cDNA molecules flanked by 3′single-stranded extensions that are complementary to the 3′single-stranded extensions on the recipient molecule:

(a) the mRNA for generating a cDNA and Primer 1 for first strandsynthesis. Primer 1 includes a priming sequence of polyT and anadditional hexaguanine sequence at its 5′ end, where guanine at the 6thposition from the 5′ end is replaced by an 8-oxo-guanine: “V”=A, C or G;“N”=A, T, C or G; and “G=O” indicates an 8-oxo-guanine;

(b) a cDNA/mRNA hybrid is generated in the presence of a reversetranscriptase (M-MuLV) and Primer 1;

(c) the mRNA is removed from the hybrid by RNase H digestion leavingsingle-stranded cDNA molecule;

(d) a poly dC tail is added at the 3′ end of the single-stranded cDNAusing a terminal transferase and dCTP. Primer 2 which includes poly dGsequence that hybridizes to the poly dC tail of cDNA is designed to havean 8-oxo-guanine at the 6th position from the 5′ end. “H”=A, T or C;

(e) the double-stranded cDNA is generated using DNA Polymerase I andPrimer 2;

(f) the double-stranded cDNA is treated with USER™ Enzyme 2 to nick the8-oxo-guanine yielding 3′ single-stranded extensions of 6 cytosines;

(g) the recipient molecules having 3′ single-stranded extensions of 6guanines are annealed to the cDNA molecules produced in (f) to formrecombinant molecules thus generating a cDNA library.

FIG. 19 shows the design of a recipient molecule pNEB205A (New EnglandBiolabs, Inc., Beverly, Mass.) and generation of a linearized vector forcloning of target molecules:

(a) the recipient molecule is here shown to be a circular vectorreferred to as pNEB205A which is constructed by inserting a cassette(SEQ ID NO:1) which contains two inversely-oriented nicking N.BbvCIBsites flanking the XbaI restriction site into the multiple cloning siteof pNEB193 vector (New England Biolabs, Catalog, 2002-2003, p. 318). Theenzyme recognition sites within the cassette are underlined and thecleavage positions within the sites are indicated by the arrows.

(b) the product (SEQ ID NO:2) of digestion of pNEB205A with N.BbvCIB andXbaI is a linear vector pNEB205A flanked with 8-nucleotide long 3′single-stranded extensions, GGGAAAGT-3′ and GGAGACAT-3′, respectively.

FIG. 20 shows the design of a recipient molecule pNEB200A (New EnglandBiolabs, Inc., Beverly, Mass.) and production of linearized vector forcloning of target molecules:

(a) the recipient molecule is here shown to be a circular vectorreferred to as pNEB200A which is constructed by inserting a cassette(SEQ ID NO:3) which contains two inversely-oriented nicking N.BstNBIsites flanking two XbaI restriction sites into the multiple cloning siteof pNEB193 vector. The enzyme recognition sites within the cassette areunderlined and the cleavage positions are indicated by the arrows.N.BstNBI cleaves outside the recognition sequences.

(b) the product of digestion of pNEB200A with N.BstNBI and XbaI is alinear vector pNEB200A flanked with 8-nucleotide long 3′ single-strandedextensions, ACGAGACT-3′ and ACCAGACT-3′, respectively.

FIG. 21 shows the design of a recipient molecule pNEB210A (New EnglandBiolabs, Inc., Beverly, Mass.) and production of linearized vector forcloning of target molecules:

(a) the recipient molecule is here shown to be a circular vectorreferred to as pNEB210A which is constructed by inserting a cassette(SEQ ID NO:4) which contains two inversely-oriented nicking N.BbvCIBsites flanking BamHI and XbaI restriction sites into the multiplecloning site of pNEB193 vector. The enzyme recognition sites within thecassette are underlined and the cleavage positions within the sites areindicated by the arrows.

(b) the product (SEQ ID NO: 5) of digestion of pNEB210A with N.BbvCIB,BamHI and XbaI is a linear vector pNEB210A flanked with 6-nucleotidelong 3′ single-stranded extension of GGGGGG-3′ on one end and8-nucleotide long 3′ single-stranded extension of GGAGACAT-3′ on theother end.

FIG. 22 shows the design of a recipient molecule pUC-TT (New EnglandBiolabs, Inc., Beverly, Mass.) and production of linearized vector forcloning of target molecules:

(a) the recipient molecule is here shown to be a circular vectorreferred to as pUC-TT which is constructed by inserting a cassette (SEQID NO:6) which contains two inversely-oriented nicking N.BbvCIB sitesflanking two BamHI restriction sites into the multiple cloning site ofpNEB193 vector The enzyme recognition sites within the cassette areunderlined and the cleavage positions within the sites are indicated bythe arrows.

(b) the product (SEQ ID NO:7) of digestion of pUC-TT with N.BbvCIB andBamHI is a linear vector pUC-TT flanked with 6-nucleotide long 3′single-stranded extensions of GGGGGG-3′ on both ends.

FIG. 23 shows the sequence (SEQ ID NO:8) of a 34-bp oligonucleotideduplex used to assay the activity of artificial nicking agents. The topstrand of duplex is fluorescently labeled on both 5′ and 3′ ends (*) andcontains a single deoxyuridine (U) at the 16^(th) position. The bottomstrand of hetero-duplex contains a deoxyadenine across from the positioncorresponding to dU.

FIG. 24 shows how the optimal amount of EndoVIII glycosylase/AP lyasecan be determined in a mixture with UDG glycosylase in order to producethe artificial nicking agent referred to as the USER™ Enzyme. The assayutilizes a substrate having a sequence shown in FIG. 23. 2-fold seriallydiluted amounts of EndoVIII varying in the range from 250 ng to 3.9 ngwere pre-mixed with 0.2 unit of UDG glycosylase and assayed for completenicking of 10 pmol of substrate. Lanes 1 is a control showing thesubstrate without enzyme treatment. Neither UDG alone (shown in lane 2)nor EndoVIII alone (shown in lane 3) is capable of nicking substratecontaining deoxyuridine, but the mixtures containing 0.2 units of UDGand at least 31.25 ng of EndoVIII yield complete nicking of 10 pmol ofsubstrate (lanes 4-7). Mixtures containing less than 31.25 ng ofEndoVIII are only partially digested (lanes 8-10).

FIG. 25 shows how the optimal amount of FPG glycosylase/AP lyase can bedetermined in a mixture with UDG glycosylase in order to produce theartificial nicking agent referred to as the USER™ Enzyme 2. The assayutilizes a substrate having a sequence shown in FIG. 23. 2-fold seriallydiluted amounts of FPG varying in the range from 4300 ng to 19 ng werepre-mixed with 0.1 unit of UDG glycosylase and assayed for completenicking of 10 pmol of substrate. Lane 1 is a control showing thesubstrate without enzyme treatment. Neither UDG alone (shown in lane 2)nor FPG alone (shown in lane 3) is capable of nicking substratecontaining deoxyuridine (U), but the mixtures containing 0.1 unit of UDGand at least 290 ng of FPG yield complete nicking of 10 pmol ofsubstrate (lanes 4-8). Mixtures containing less than 290 ng of FPGglycosylase/AP lyase are only partially digested (lanes 9-12).

FIG. 26A shows a design strategy for primers suitable for use inamplifying target molecule in order to generate 3′ single-strandedextensions complementary with the 3′ extensions on the linearized vectorpNEB205A prepared as shown in FIG. 19. The left primer at the 5′ endinclude sequence GGAGACAU (SEQ ID NO:9) which is identical to the rightextension on the pNEB205A in FIG. 19, except for 3′-terminal thyminewhich is replaced by deoxyuridine (U) in the primer sequence followed bythe target molecule-specific sequence from the 5′ end. The right primerat 5′ end include sequence GGGAAAGU (SEQ ID NO:10) which is identical tothe left extension on the pNEB205A in FIG. 19, except for 3′-terminalthymine which is replaced by deoxyuridine (U) in the primer sequencefollowed by the 3′ terminal target molecule-specific sequence from thereverse strand.

FIG. 26B shows an overview of target molecule cloning method:

(a) a left primer (SEQ ID NO:11) and a right primer for amplifyingtarget DNA were designed according to FIG. 26A;

(b) the amplified DNA includes a target molecule sequence which isextended at both ends by vector compatible sequences. A single U occursat each junction and on the opposite strands with respect to each other;

(c) the amplified target molecule having 3′ single-stranded extensionsgenerated after nicking at deoxyuridine (U) with the USER™ Enzyme anddissociation of the nicked 5′-terminal strand;

(d) linear vector pNE205A is shown in an inverted orientation relativeto FIG. 19, having 3′ single-stranded extensions complementary tosingle-stranded extensions on the target molecule in step (c);

(e) the recombinant molecule resulting from combining the targetmolecule and the linearized vector pNEB205A of steps (c) and (d).

FIG. 27A shows a restriction map of the pNEB205A plasmid. pNEB205A isidentical to either pNEB193 (New England Biolabs Catalog 2002-2003, p.318) or pUC19 (Yanisch-Perron, et al. Gene 33:103-119 (1985)) except forthe multiple cloning site (MCS). The new MCS is in frame with the lacZαgene, allowing screening for insertions using α-complementation.

FIG. 27B shows the nucleotide sequence (SEQ ID NO:12) of the multiplecloning site (MCS) of pNEB205A. The nucleotide sequence is numbered toshow the location of the MCS within pNEB205A. The N-terminal amino acidsequence of the LacZα fragment is shown under the respective codons.Restriction sites are underlined. Cleavage sites of XbaI restrictionendonuclease and N.BbvCIB nicking endonuclease are shown as blacktriangles. The shaded area shows the nucleotide sequence which isremoved from pNEB205A after digestion with XbaI and N.BbvCIB.

FIG. 27C shows the sequence (SEQ ID NO:13 and SEQ ID NO:14) of 3′single-stranded extensions on the linearized pNEB205A vector, which werecreated by digestion with N.BbvCIB and XbaI within the MCS shown in FIG.27B.

FIG. 28 shows the PCR-amplified Chloramphenicol Resistance gene (cat)DNA (0.95 kb) in a 10 μl of amplification sample (out of 50 μl of thetotal PCR volume). Lanes 1 and 12 contain a 2-Log DNA Ladder (NewEngland Biolabs, Inc., Beverly, Mass.). Lanes from 2 to 10 show PCRsample after 8, 9, 10, 11, 13, 16, 20, 25 and 30 cycles containing 5,10, 17, 45, 82, 164, 215, 346 and 390 ng of DNA, respectively. Lane 11shows 1 μl (20 ng) of the linearized vector pNEB205A.

FIG. 29 shows the number of colonies produced by 25 μl of transformationreaction. White colonies represent transformants carrying recombinantmolecules. Blue colonies represent transformants carrying unmodifiedvector. Different amounts of PCR product varying in the range from 5 ng(0.0008 pmol) to 390 ng (0.62 pmol) were assembled into 20 ng (0.011pmol) of linear pNEB205A following the reaction protocol described inExample III. The transformation results represent the average of threeindependent experiments. (±) indicates the standard deviation value (s)which was calculated using the formula: s²=S(X−M)²/(N−1), where X is themeasurement value, M is the mean and N is the number of measurements.Cloning efficiency was determined by calculating the fraction of whitecolonies in the total number of transformants.

FIG. 30 shows the graphical illustration of the results presented inFIG. 29 showing that fraction of recombinants is 94%-95% if theconcentration of PCR product is 50 ng (0.08 pmol) or higher.

FIG. 31 shows the design strategy of primer pairs P2/P3 and P1/P4 forgenerating intermediate PCR fragments of a target molecule flanked withcomplementary 3′ single-stranded extensions. The overlapping Primers P1and P2 start with a 5′ adenine; (N)₁₋₂₀ indicate the overlap regionshared by overlapping primers where each primer contains the reversecomplement sequence of the other. The overlap sequence at the 3′ end isflanked by a deoxyuridine (U), which is across from the 5′ adenine onthe opposite primer. Downstream of deoxyuridine (U), the primers primethe respective sequences on the target molecule.

FIG. 32 shows site-specific mutagenesis in which a codon substitution (3nucleotide substitution) is introduced into a gene encoding HincIIrestriction endonuclease (SEQ ID NO:16):

(a) design of primer pairs P2/P3 and P1/P4 for amplification of hincIIRgene as two overlapping fragments. To create overlap region, a9-nucleotide sequence which starts with adenine and ends with thymine(marked as boxed area) is selected on the sequence of hincIIR gene inthe vicinity of nucleotides targeted for mutagenesis (marked byasterisks). This sequence is included at the 5′ ends of the overlappingPrimers P1 and P2, except that deoxyuridine (U) is introduced at thelast position of the overlap sequence. In Primer P1 downstream of U,codon CAA is replaced by codon TTT followed by the hincIIR-specificsequence for priming. The Primer P2 sequence downstream of U iscomplementary to hincIIR-specific sequence. NdeI site is engineered at5′ end of Primer P3 and SapI site is engineered at 5′ end of Primer P4;

(b) two PCR amplification fragments are shown. Both fragments overlap bya 9-bp sequence and contain a single uracil residue on the oppositestrands. Left-side fragment contains codon TTT instead of a wild typecodon CAA.

(c) the fragments flanked by single-stranded extensions that arecomplementary to each other produced after nicking at the uracils withthe USER™ Enzyme;

(d) the fragments are annealed and ligated with T4 DNA Ligase throughtheir complementary extensions to form the modified nucleotide sequence(SEQ ID NO:18) of hincIIR gene.

(e) agarose gel electrophoresis showing the results of the pilotligation. Lanes 1 and 5 show 2-Log DNA Ladder. Lane 2 shows 1 μl of PCRsample containing a 420-bp hincIIR gene fragment. Lane 3 shows 1 μl ofPCR sample containing a 380-bp hincIIR gene fragment. Lane 4 shows 10 μlof the pilot ligation reaction containing the 780-bp hincIIR gene withthe codon CAA substitution to codon TTT.

FIG. 33 shows the insertion of two unique restriction sites and deletionof 18-bp sequence from pUC19 plasmid:

(a) the primer pairs P1/P4 and P2/P3 are designed according to FIGS. 10Aand 11 as follows. The overlapping Primers P1 and P2 at their 5′ endscontain an additional 6-nucleotide insertion sequences required forcreation of BsrGI and AvrII restriction sites. The insertion sequencesin both primers are complementary to each other and contain deoxyuridine(U) at the 6th position. Downstream of U, Primers P1 and P2 prime therespective pUC19 sequences, which start precisely beyond the targeted 18bp deletion region (SEQ ID NO:19). Primer P3 primes pUC19 sequenceacross the BsaI site and Primer P4 primes pUC19 sequence across theHindIII site;

(b) two PCR amplification fragments with a 6-bp overlapping sequencewhere each fragment has a single uracil residue positioned on theopposite strand with respect to each other;

(c) the PCR fragments flanked by single-stranded extensions that arecomplementary to each other formed after nicking at the uracils with theUSER™ Enzyme;

(d) the product (SEQ ID NO:20) of annealing and ligation of fragmentswith T4 DNA Ligase through complementary single-stranded extensions. Theproduct contains the recognition sites of restriction endonucleasesBsrG1 and AvrII;

(e) agarose gel electrophoresis showing the results of ligation. Lane 1shows 2-Log DNA Ladder. Lane 2 shows 610-bp and 810-bp PCR fragements. 1μl of each PCR sample was combined and loaded on gel. Lane 3 shows 10 μlof the ligation reaction containing the 1420-bp ligated pUC19 fragment.Lane 4 shows the ligated 1420-bp fragment is cleaved with BsrGIrestriction endonuclease. Lane 5 shows the ligated 1420-bp fragment iscleaved with AvrII restriction endonuclease.

FIG. 34 shows fusion of genes coding for E. coli Endonuclease VIII andMxe Intein:

(a) the primer pairs P1/P4 and P2/P3 are designed according to FIG. 12as follows. Overlapping Primers P1 and P2 prime the respectivegene-specific sequences and at their 5′ ends have a 7-bp overlappingsequence flanked by deoxyuridine (U). To create the 7-bp overlap, PrimerP2 at its 5′ end is extended by five 5′-terminal nucleotides of the MxeIntein gene, and Primer P1 at its 5′ end is extended by two 3′-terminalnucleotides of EndoVIII gene. An NdeI site is engineered at 5′ end ofPrimer P3 and Primer P4 primes across the AatII site in Mxe Intein gene;

(b) two PCR amplification fragments having the common overlap region ofseven nucleotides as described in (a). Both amplification productscontain a single uracil residue positioned on the opposite strands withrespect to each other;

(c) the fragments flanked by single-stranded extensions that arecomplementary to each other produced after nicking at the uracils withthe USER™ enzyme;

(d) the product of annealing and ligation of fragments with T4 DNALigase through complementary single-stranded extensions. The product isa precise fusion of two genes.

(e) agarose gel electrophoresis showing the results of the ligation. 1μl PCR samples containing 265-bp fragment of the Mxe Intein gene and an800-bp EndoVIII gene were combined and loaded on gel (Lane 1). Lane 2shows 2 μl of the ligation reaction showing the 1065-bp ligationproduct, which represents a Mxe Intein gene fragment fused to the 3′ endof the EndoVIII gene. Lane 3 shows 2-Log DNA Ladder.

FIG. 35 shows concurrent site-specific mutagenesis and gene fusion:

(a) shows the final product of site-specific mutagenesis and genefusion. A hincIIR gene sequence is altered to generate hincIIQ138Avariant, and inserted between the promoter region of pTXB1 (SEQ IDNO:21) (New England Biolabs, Inc., Beverly, Mass.) vector and the 5′ endof the Mxe Intein gene. The start positions of hincIIR and Mxe Inteingenes are indicated by arrows. Asterisks indicate the position ofnucleotides altered in the primer sequences to introduce a codon CAA toGCT substitution. The nucleotide sequences of the overlap regions usedto design overlapping primer pairs P2/P3, P4/P5 and P6/P7, are shownwithin the boxed area.

(b) primer pairs P1/P2, P3/P4, P5/P6 and P7/P8 were designed for DNAamplification according to FIG. 13. The overlapping primer pairs P2/P3(SEQ ID NO:22), P4/P5 and P6/P7 have at their 5′ ends the overlappingsequences shown within the respective boxed area in (a) and contain adeoxyuridine (U) at the 3′-terminal position of the overlap sequence.Primer P1 primes across the XbaI site in the promoter region of pTXB1vector and Primer P8 primes across the BsrGI site in the Mxe Inteingene;

(c) four PCR amplification fragments each having a common overlap regionwith the next in line fragment. The fragment coding for pTXB1 promoterregion overlaps by 11-bp sequence with the 5′ terminal part of thehincIIR gene; the 5′ terminal part of hincIIR gene overlaps by 9-bpsequence with the 3′-terminal part of the hincIIR gene; and the3′-terminal part of the hincIIR gene overlaps by 9-bp sequence with the5′-terminal part of the Mxe Intein gene. The hincIIR fragments at thejunction have a desired nucleotide changes (indicated by asterisks). Theoutside amplification products contain a single uracil residuepositioned on the opposite strands, while two middle fragments thatcontain two uracils per fragment positioned on the opposite strands;

(d) the fragments flanked by single-stranded extensions that arecomplementary to each other after nicking at uracils with the USER™enzyme;

(e) the product of annealing and ligation of fragments with T4 DNALigase through complementary single-stranded extensions. The product isa precise fusion of four fragments, which is identical to (a);

(f) agarose gel electrophoresis showing the results of the pilotligation. Lane 1 shows 1 μl of PCR sample containing a 140-bp fragmentof the promoter region of pTXB1 vector. Lane 2 shows 1 μl of PCR samplecontaining a 420-bp fragment of the hincII gene. Lane 3 shows 1 μl ofPCR sample containing a 380-bp fragment of the hincII gene. Lane 4 shows1 μl of PCR sample containing a 360-bp fragment of the 5′ terminal MxeIntein gene. Lane 5 shows 10 μl of the pilot ligation reaction with the1270-bp ligation product, which represents the linearly assembled finalconstruct. Lane 6 shows 2-Log DNA Ladder.

FIG. 36 shows the assembly of human AP1 (hAP1) endonuclease gene fromtotal human genomic DNA concurrent with site-specific mutagenesis:

(a) cDNA of the hAP1 endonuclease gene with Exons 2-5. The junctions ofthe individual exons are shown by vertical dotted lines. Exon 5 containsthe NdeI site which is targeted for silent mutation of A to G (indicatedby asterisk). The nucleotide sequences of the overlap regions, whichwere used to design overlapping primer pairs P2/P3, P4/P5, P6/P7 andP8/P9, are shown within the boxed area.

(b) primer pairs P1/P2, P3/P4, P5/P6, P7/P8 and P9/P10 for amplificationof individual exons of hAP1 gene from total genomic DNA were designed asfollows. Overlapping primers P2/P3, P4/P5, P6/P7 and P8/P9 haveoverlapping sequences at their 5′ ends shown within the boxed area in(a), and further containing a deoxyuridine (U) at the 3′-terminalposition of the overlap sequence. An NdeI site is engineered at 5′ endof Primer P1 and SapI site is engineered at 5′ end of Primer P10;

(c) five PCR amplification fragments each having the common overlapregion with the adjacent fragment. The 3′ end of Exon 2 overlaps withthe 5′ end of Exon 3 (SEQ ID NO:23); the 3′ end of Exon 3 overlaps withthe 5′ end of Exon 4; the 3′ end Exon 4 overlaps with the 5′ end of thefirst part of Exon 5 which on its 3′ end overlaps with the 5′ end of thesecond part of Exon 5. The overlapping fragments of Exon 5 at thejunction have a desired nucleotide change (indicated by asterisk). Theoutside amplification products contain a single uracil residuepositioned on opposite strands, while the three middle fragments containtwo uracils per fragment on opposite strands.

(d) the fragments flanked by single-stranded extensions that arecomplementary to each other in the specified order after nicking aturacils with the USER™ Enzyme;

(e) the product of annealing and ligation of fragments with T4 DNALigase through complementary single-stranded extensions. The product isa precise and ordered fusion of five fragments constituting the hAP1gene with the mutated Exon 5 sequence;

(f) agarose gel electrophoresis showing the results of the pilotligation. Lanes 1 and 8 show 2-Log DNA Ladder. Lane 2 shows 1 μl of PCRsample containing an 80-bp fragment of the Exon 2. Lane 3 shows 1 μl ofPCR sample containing a 180-bp fragment of the Exon 3. Lane 4 shows 1 μlof PCR sample containing a 200-bp fragment of the Exon 4. Lane 5 shows 1μl of PCR sample containing an 80-bp fragment of the 5′ terminal portionof the Exon 5. Lane 6 shows 1 μl of PCR sample containing a 460-bpfragment of the 3′ terminal portion of the Exon 5. Lane 7 shows 10 μl ofthe pilot ligation reaction with the 1000-bp ligation product, whichrepresents the hAP1 gene.

FIG. 37 shows directional assembly of mutagenized fragments of 9° N_(m)DNA Polymerase gene (SEQ ID NO:25) into the linearized pNEB205A vector:

(a) primer pairs P1/P4 and P2/P3 were designed according to FIG. 16 asfollows. The nucleotide sequence of the overlap region, which was usedto design the overlapping Primers P1 and P2, is shown within the boxedarea. “*” indicates the positions of nucleotides that were changed inthe Primer P1 (SEQ ID NO:24) and P2 sequences to introduce codon GTC toCAA substitution in the 9° N_(m) DNA Polymerase gene. Primers P3 and P4on their 5′ ends were supplemented with the additional sequences thatwere compatible with the 3′ single-stranded extensions on the pNEB205Avector;

(b) two PCR amplification fragments (SEQ ID NO:26 and SEQ ID NO:24)having a common overlap region of ten nucleotides. Within the overlapregion, the fragments have desired nucleotide changes (indicated byasterisks). Each fragment at the outside end is extended by eightnucleotides complementary to the corresponding extensions on pNEB205Ashown in step (d). Both amplification products contain two uracilresidues positioned on each end of fragment and on the opposite strandswith respect to each other;

(c) fragments flanked by single-stranded extensions after nicking aturacils with the USER™ enzyme;

(d) linear pNEB205A vector flanked by 3′ single-stranded extensionswhich are complementary to the outside extensions of fragments in step(c);

(e) recombinant molecule generated by annealed the fragments intopNEB205A vector through their complementary extensions.

FIG. 38 shows the cloning of unknown 3′ segment of the super-integronfrom Pseudomonas alcaligenes NEB#545 (New England Biolabs, Inc.,Beverly, Mass.) into pUC-TT vector:

(a) Primer Pal3-1 primes the super-integron sequence in contig C forproducing single-stranded polynucleotide molecules of different lengthscontaining the unknown sequence of super-integron;

(b) polyC tails were added to the 3′ ends of the amplifiedsingle-stranded molecules using terminal transferase and dCTP. PolyCtailed single-stranded fragments were then amplified with Primers Pal3-3(SEQ ID NO:27) and GG-2. Primer Pal3-3 primes specific super-integronsequence approximately 60 nucleotides from the 3′ end and containshexaguanine sequence at 5′ end with an 8-oxo-guanine at the 6^(th)position. Primer GG-2 is poly-dG, which is complementary to the polyCtail and contains an 8-oxo-guanine at the 6^(th) position from 5′ end.“N”—A, G, C or T; “H”—indicates A, T or C;

(c) double-stranded amplification products flanked by hexaguaninesequences on both ends and containing two 8-oxo-guanine residues permolecule which are positioned on opposite strands with respect to eachother;

(d) amplified products after nicking with FPG glycosylate/AP lyase at8-oxo-guanine to generate 3′ single-stranded extensions of six cytosineson both ends of the amplified products;

(e) the amplified products representing the unknown sequences ofsuper-integron are inserted into the linear pUC-TT vector having 3′single-stranded extensions of six guanines thus creating recombinantmolecules.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms as used in the Description and in the accompanyingclaims have been defined below. These definitions should be appliedunless the context in which the terms are used requires otherwise.

The term “single-stranded extension” as used herein refers to asingle-stranded region extending from a double-stranded region of apolynucleotide molecule.

The term “polynucleotide molecule” refers to single-stranded ordouble-stranded DNA molecule or RNA molecule or an RNA/DNA hybrid. Thepolynucleotide molecule may be of any length and includesoligonucleotides, plasmids, chromosomal DNA, double-stranded RNA,mRNA/DNA hybrids, amplified DNA fragments and other naturally occurringor synthetic double-stranded nucleic acids.

The term “primer” refers to an oligonucleotide sequence which forms asubstrate for polymerase-dependent amplification of a target moleculewhere at least part of the oligonucleotide sequence is complementary toa pre-selected sequence on one strand of a double-stranded targetpolynucleotide molecule. The oligonucleotide sequence may be preparedsynthetically using standard techniques.

The term “target molecule” as used herein refers to a portion of apolynucleotide molecule, for example DNA, which is selected formanipulation.

The term “recipient molecule” as used herein includes a double-strandedpolynucleotide molecule capable of being replicated in a host cell.

The term “linearized vector” as used herein refers to a recipientmolecule that is converted into linear form or to any other non-circularDNA.

The term “recombinant molecule” as used herein refers to apolynucleotide molecule that is composed of at least two targetmolecules. The term also includes a circular polynucleotide moleculethat is composed of a recipient molecule and at least one targetmolecule so that the replication of the inserted target molecule(s) inthe recipient molecule may occur in a host cell.

The term “host cell” refers to any eukaryotic or prokaryotic cell,including cells from mammals, insects, yeast, bacteria or otherorganisms without limit.

The term “specific nicking” refers to hydrolysis of a phosphodiesterbond within a polynucleotide molecule at the selected location. Afterspecific nicking, the polynucleotide molecule is no longer continuouslycovalently linked at the selected location.

The term “selected location” refers to a specific sequence in apolynucleotide molecule or primer that contains at least one nucleotide,which may be a modified nucleotide.

The term “cassette” refers to a double-stranded nucleic acid. Thecassette contains a pre-selected combination of at least onesequence-specific nicking site recognized by a sequence-specific nickingendonuclease and at least one sequence-specific restriction siterecognized by a sequence-specific restriction endonuclease. The nickingand restriction sites in the cassette are separated from each other by adefined sequence and are ordered and oriented with respect to eachother. The boundaries of the cassette in its minimum configuration aredetermined by the position of the nicking site and a restriction site orby the position of two nicking sites as shown in FIGS. 19-22. However,the boundaries of the cassette can be extended to incorporatenon-essential nucleotide sequences outside the minimum configuration.

The term “modified nucleotide” refers to a nucleotide that is chemicallydistinguishable from unmodified nucleotides that occur in nature namelydA, dT, dG and dC. The modified nucleotides are further characterized bytheir ability to be incorporated in polynucleotide molecules in theplace of unmodified nucleotide.

The term “nicking agent” refers to a reagent which is capable of bothrecognizing a sequence-specific target, and nicking the target at aphosphodiester bond within or in a defined relationship to suchsequence-specific target. The target comprises at least one nucleotide,where the nucleotide is a modified nucleotide.

The term “artificial” refers to reagents or molecules that have beencombined in vitro to achieve a particular purpose.

The use of the term “include” is intended to be non-limiting.

The term “DNA glycosylase” refers to any enzyme with glycosylaseactivity which causes excision of a modified nitrogenous heterocycliccomponent of a nucleotide from a polynucleotide molecule.

The term “single-strand cleavage enzyme” refers to (i) an APendonuclease, lyase or other enzyme which cleaves a phosphodiester bondafter an AP site is formed, or (ii) an enzyme that cleave directly at amodified nucleotide or a single-stranded region in a polynucleotidemolecule.

Described below are methods and compositions relating to generation ofsingle-stranded extensions of defined length and composition inpolynucleotide molecules. Any molecular biology technique for which aspecific reference has not been provided herein may be achievedfollowing the protocols provided in Sambrook, In Molecular Cloning.Laboratory Manual 2001 (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

Single-stranded extensions created on polynucleotide molecules accordingto present embodiments of the present invention are characterized bytheir precise design with respect to their length and nucleotidesequence. The single-stranded extension on a polynucleotide molecule canbe of any desired length with no particular limitation on the maximumlength that can be produced according to embodiments of the methodsdescribed here. However, in practice, the length of the single-strandedextension is selected according to its ability to self-dissociate from acomplementary strand. Consequently, the preferred length of asingle-stranded extension is no greater than about 20 nucleotides, andpreferably less than about 14 nucleotides. For the uses describedherein, the length of the single-stranded extension should be at leastabout 5 nucleotides.

Creating Single-Stranded Extensions Using Cassettes

A process of generating a single-stranded extension in a polynucleotidemolecule involves first inserting a cassette into a polynucleotidemolecule. The cassette can be inserted into any polynucleotide moleculesusing, for example, restriction endonuclease cloning method or PCRamplification.

Cleavage of a polynucleotide molecule containing the cassette with theselected combination of nicking endonuclease(s) and restrictionendonuclease(s) produces at least one single-stranded extension ofdesired length and composition. The one or more nicking endonucleasesspecifically nick the cassette on one nucleic acid strand only. The oneor more restriction endonucleases cleave the cassette on both nucleicacid strands. Within the cassette, the location and orientation of theone or more nicking site with respect to the one or more restrictionsites determines the orientation and the length of the single-strandedextensions. The length may be changed at will by incorporating aselected number of nucleotides in between the nicking site and therestriction site.

The nucleotide composition of the single-stranded extensions partiallydepends on the nucleotide sequence of the nicking and restriction sitespresent in the cassette. A significant contribution to the compositionof the single-stranded extension comes from the variable nucleotidesequence incorporated in between each nicking site and restriction site.In practice, the practitioner chooses the most convenient nicking andrestriction sites from those sites known in the art for incorporationinto the cassette and customizes the length and composition of thenucleotide sequences in between the sites to obtain single-strandedextensions with the desired nucleotide composition.

The cassette can be designed so that a single-stranded extension may begenerated on one or both ends of a cleaved polynucleotide molecule andon either the 3′ or 5′ terminus of the duplex. This can be achieved byselective orientation of the nicking sites with respect to therestriction site in the cassette. Since nicking endonucleases cleaveonly one strand at a double-stranded nicking site, the orientation ofthe site determines whether the top or the bottom strand is nicked. Theterminal sequence flanked by the nick self-dissociates from thecomplementary strand because the nick is introduced close to thedouble-strand break caused by restriction endonuclease. Afterself-dissociation, the polynucleotide molecule is left flanked by eithera 5′ or 3′ single-stranded extension according to which strand isnicked.

The design of cassette for generation of single-stranded extensions on apolynucleotide molecule is flexible. The flexibility arises from theoptions available for arranging the order, orientation and spacing ofthe nicking site(s) with respect to adjacent restriction site(s). Thepreferred length of the cassette is greater than 5 nucleotides. Where aspacer region is present between two restriction sites, there is noupper limit on the length of the cassette. In the presence of onerestriction site only, the upper limit on the length of the cassette isdetermined by the ability of the terminal nicked strand toself-dissociate from the un-nicked strand.

What has been generally described above is here described in morespecific detail. Optional configurations of a cassette are exemplifiedin FIGS. 1-6.

FIGS. 1, 2 and 5 show how the cassettes may be designed to produce 3′ or5′ single-stranded extension on a selected end of a polynucleotidemolecule. FIG. 1 depicts a cassette where the nicking site is upstreamof the restriction site and in an orientation that allows nicking of thebottom strand. Double-digestion of a polynucleotide molecule containingthe cassette with restriction and nicking endonucleases generates a 3′single-stranded extension on the left-side fragment of polynucleotidemolecule. Alternatively, a 3′ single-stranded extension on theright-side fragment of polynucleotide molecule can be generated, whenthe nicking site is placed downstream of the restriction site and isinverted to provide top-strand nicking (FIG. 2). Similarly, a 5′single-stranded extension can be generated on the right or left fragment(FIG. 5).

FIGS. 3, 4 and 6 show how cassettes can be designed to generatesingle-stranded extensions of desired length and composition on bothends of a polynucleotide molecule. Accordingly, the cassette includestwo inversely-oriented nicking sites located on either side of a singlerestriction site (FIG. 3) or on either side of two restriction siteswhere these sites may be the same (FIG. 4) or different (FIG. 6) toyield two single-stranded extensions. The cassettes depicted in FIGS. 3and 4 show two nicking sites of the same nicking endonuclease: Site Aand inverted Site A both are recognized by nicking endonuclease A andopposite strands are nicked because of the inverted orientation of thesites. It is also possible to use of two non-identical nicking sitesrecognized and nicked by two distinct nicking endonucleases. Similarly,the cassettes in FIGS. 4 and 6 may include two non-identical restrictionsites assuming that the recognition sequences provide the desirednucleotide composition for generation of single-stranded extensions. Forexample, FIGS. 5 and 6 depicts cassettes containing nicking site(s) fora nicking endonuclease C and a restriction site for restrictionendonuclease D whereas in FIGS. 1 to 4, sites A and B are shown.

A cassette such as that shown in FIG. 3 may be modified to include anadditional spacer sequence (FIG. 6). Cleavage with the restrictionendonuclease and nicking endonuclease results in removal of the spacersequence and generating single-stranded extensions identical to those inFIG. 3. An advantage of introducing a spacer sequence into the cassetteis to provide a nucleotide sequence which codes for a selectable marker.The presence of the selectable marker enables differentiation betweencleaved and non-cleaved cassettes in a transformed host cell. Examplesof selectable markers include toxins, drug-resistant factors, enzymes,antigens, fluorescent and chemiluminescent markers, and siRNA.

Nicking sites include any sequences that are specifically recognized andnicked by nicking endonuclease. The sequence of the nicking siteencompasses the sequence at which the endonuclease binds and thesequence at which it cleaves, be it within the recognition site oroutside the recognition site. Any nicking endonuclease which nickswithin its recognition site or at a distance from their recognition sitemay be utilized. Examples include nicking endonuclease N.BstNBI whichrecognizes and nicks GAGTCNNNN↓; N.BbvCIB which recognizes and nicksCC↓TCAGC; N.BbvCIA which recognizes and nicks GC↓TGAGG; N.AlwI whichrecognizes and nicks GGATCNNNN←; and N.Bpu10I which recognizes and nicksGC←TNAGG (U.S. Pat. No. 6,395,523 and EPO Grant No. 1 176 204). Includedhere is the use of any nicking endonuclease derived from modification ofa Type II restriction endonuclease, for example, by methods discussed inInternational Application No. PCT/US02/26273 and U.S. application Ser.No. 09/738,444.

Restriction sites include any sequences that are specifically recognizedby restriction endonucleases. The restriction site encompasses thesequence at which the endonuclease binds and the sequence at which itcleaves be it within the recognition site or outside the recognitionsite. Examples of restriction endonucleases include any of therestriction endonucleases listed in REBASE® (www.NEB.com).

The polynucleotide molecules for which single-stranded extensions ofdesired length and composition may be created include recipientmolecules which further include circular vectors and linear moleculessuch as genomic DNA or fragments of DNA. Using a single-strandedextension on a polynucleotide molecule which is complementary to asingle-stranded extension on another polynucleotide molecule, two ormore polynucleotide molecules may be joined to form a single molecule.For example, a target DNA molecule may be inserted into a recipientmolecule using complementary single-stranded extensions.

Single-stranded extensions on the recipient molecule may be created byfirst inserting a cassette into the recipient molecule. The cassette issubsequently cleaved with an appropriate combination of restrictionendonuclease(s) and nicking endonuclease(s) to generate a linearizedrecipient molecule having single-stranded extensions of desired lengthand composition on both ends.

Recipient molecules with 3′ or 5′ single-stranded extensions permitinsertion of polynucleotide molecules (target molecules) which havecomplementary single-stranded extensions to form recombinant molecules.The recombinant molecules can be replicated in a transformed host cell.

The generation of linearized recipient molecules with 3′ single-strandedextensions of desired length and composition is simple to perform andhas advantages which include: (a) single-stranded extensions may be ofany desired length, i.e. longer than those produced by cleavage withrestriction endonuclease; (b) single-stranded extensions can be designedto be non-self-complementary, that is they are not complementary to eachother, so that recipient molecule termini do not re-anneal to formtransformable circular DNA; and (c) each single-stranded extension maybe designed to carry a unique nucleotide sequence, thereby permittingcontrol of the orientation of the inserted target molecule. Examples ofrecipient molecules containing cassettes designed to create different 3′single-stranded extensions are provided in Example I and FIGS. 19-22 andFIG. 27 and include plasmid vectors pNEB200A, pNEB205A, pNEB210A andpUC-TT (New England Biolabs, Inc., Beverly, Mass.).

In particular, the cassette in vector pNEB205A (FIG. 19) carries twoN.BbvCIB nicking sites, which are separated by a single XbaI restrictionsite. The N.BbvCIB and XbaI sites are arranged in such a way thatdigestion of the recipient molecule with the above specified enzymesprovides a linearized vector flanked by 8-nucleotide longsingle-stranded extensions, GGGAAAGT-3′ and GGAGACAT-3′, on the ends.The cassette in vector pNEB200A (FIG. 20) carries two N.BstNBI nickingsites, which are separated by two XbaI restriction sites. The N.BstNBIand XbaI sites are arranged in such a way that digestion of therecipient molecule with the above specified enzymes provides alinearized vector flanked by 8-nucleotide long single-strandedextensions, ACGAGACT-3′ and ACCAGACT-3′, on the ends. The cassette invector pNEB210A (FIG. 21) carries two N.BbvCIB nicking sites, which areseparated by XbaI and BamHI restriction sites. The N.BbvCIB, XbaI andBamHI sites are arranged in such a way that digestion of the recipientmolecule with the above specified enzymes provides a linearized vectorflanked by 6-nucleotide long single-stranded extension of GGGGGG-3′ onone end and 8-nucleotide long single-stranded extension of GGAGACAT-3′on the other end. The cassette in vector pUC-TT (FIG. 22) carries twoN.BbvCIB nicking sites, which are separated by two BamHI restrictionsites. The N.BbvCIB and BamHI sites are arranged in such a way thatdigestion of the recipient molecule with the above specified enzymesprovides a linearized vector flanked by 3′ single-stranded extensions of6-guanines on both ends.

Although specific examples of cassettes are provided above forgenerating single-stranded extensions, a variety of restriction sitesand/or nicking sites can be used to generate the same single-strandedextensions. For example, restriction sites for either BspHI (T↓CATGA) orBspEI (T↓CCGGA) or BclI (T↓GATCA) or BsrGI (T↓GTACA) may substitute forthe XbaI site to create the identical single-stranded extension shown inFIG. 20. Nicking sites for N.AlwI or N.BbvCIA may be used in place ofeither N.BstNBI or N.BbvCIB sites for alternative sites. The choice ofrestriction and nicking sites is limited only by considerations of theintended use of the resultant single-stranded extensions.

Generation of Single-Stranded Extensions Using a Nicking Agent

An alternate method of generating 3′ single-stranded extensions fromthat described above involves primer dependent amplification of targetmolecules where the primers contain a modified nucleotide at a specificsite. The amplification product is treated with a nicking agent thatnicks specifically at the modified nucleotide. Dissociation of thenicked single-stranded terminal region from complementary strandgenerates a single-stranded extension. Examples of primer-dependentamplification include: Polymerase Chain Reaction (PCR), StrandDisplacement Amplification (SDA), Transcription-Mediated Amplification(TMA) and Ligase Chain Reaction (LCR).

The use of modified nucleotides incorporated into amplificationfragments provide the following advantages: (a) the length of thespecific nick site may be as short as one nucleotide; (b) the modifiednucleotide may be incorporated at the pre-selected location in targetmolecule and represents a unique target for nicking; and (c) anyindividual modified nucleotide out of a number of different types ofmodified nucleotides can be incorporated into each of several locationsin the same or several different target molecules thus creating aplurality of unique specific nicking sites.

Examples of modified nucleotides for incorporation into target moleculesinclude deoxyuridine (U), 8-oxo-guanine (8oxoG), 5,6-dihydrothymine,thymine glycol, uracil glycol, 5-hydroxymethylcytosine,5-hydroxymethyluracil, 7-methyladenine, 7-methylguanine, hypoxanthine,xanthine and others. Incorporation of modified nucleotides at theselected locations of target molecule may be achieved by means wellknown to those skilled in the art that include either chemical orenzymatic synthesis of polynucleotide molecule (Piccirilli et al. Nature343:33-37 (1990); Purmal et al. Nucl. Acid. Res. 22:72-78 (1994);Horlacher et al. Proc. Natl. Acad. Sci. USA 92:6329-6333 (1995); Kamiyaet al. Nucl. Acid. Res. 23:2893-2899 (1995); Lutz et al. Nucl. Acid.Res. 24:1308-1313 (1996); Zhang et al. Nucl. Acid. Res. 25:3969-3973(1997); Hill et al. Nucl. Acid. Res. 26:1144-1149 (1998); Liu, et al.,Nucl. Acid. Res. 26:1707-1712 (1998); Berdal et al. EMBO J. 17:363-367(1998); Purmal et al. J. Biol. Chem. 273:10026-10035 (1998); Lutz et al.Nucl. Acid. Res. 27:2792-2798 (1999); Duarte et al. Nucl. Acid. Res.27:496-502 (1999); Pourquier et al. J. Biol. Chem. 274:8516-8523 (1999);Kamiya, et al., Nucl. Acid. Res. 28:1640-1646 (2000); Duarte et al.Nucl. Acid. Res. 28:1555-1563 (2000)).

In one embodiment, a single-stranded oligonucleotide containing at leastone modified nucleotide may be synthesized in vitro by means thatinclude chemical synthesis. Chemically synthesized oligonucleotidemolecules may be used as primers for amplification of a sequence in atarget molecule. In one aspect of the embodiment, a modified nucleotidemay be incorporated into a primer sequence close to its 5′ end, forexample the modified nucleotide may be incorporated at a distance of 2to 20 nucleotides from the 5′ end. The 5′ end of primer sequenceupstream of the modified nucleotide may not necessarily anneal to thesequence of the target molecule; instead it may contain a customdesigned sequence which is complementary to the 5′ region of anotherpolynucleotide molecule. In these circumstances, the primer sequencedownstream of the modified nucleotide is complementary to a selectedregion of the target molecule allowing the enzymatic extension, copyingthe target molecule onto the 3′ end of primer sequence. Otherconsiderations of primer design, for example such as the length ofpriming sequence and the melting temperature, are well known to the art(Sambrook, J. in: Molecular Cloning. Laboratory Manual, pp. 8.13-8.16(2001) Cold Spring Harbor laboratory Press. Cold Spring Harbor, N.Y.).

Primer sequences containing at least one modified nucleotide becomeincorporated into a double-stranded target molecule otherwise lackingsuch modified nucleotides by multiple rounds of enzymatic DNA copying,for example, by primer extension, by Polymerase Chain Reaction (PCR)dependent DNA amplification or by other primer dependent amplificationmeans such as Strand Displacement Amplification (SDA). Methods andconditions for either primer extension or amplification are widely knownin the art (see for example U.S. Pat. Nos. 4,683,195; 4,683,202;5,333,675; Sambrook, J. in: Molecular Cloning. Laboratory Manual, pp.8.4-8.29 (2001) Cold Spring Harbor laboratory Press. Cold Spring Harbor,N.Y.). Preferably, the DNA polymerase utilized in the DNA copyingreaction is one that incorporates the correct nucleotide opposite themodified nucleotide in at least 50% of product molecules. Since primersequences, but not the target molecule, define the 5′ ends of theamplified molecule, the target molecule after amplification may beextended by an additional sequence at least on one end, and may carry amodified nucleotide at the junction between target sequence and theadditional sequence.

The amplified target molecule can be specifically nicked at thelocation(s) of the modified nucleotide(s) using a modifiednucleotide-specific nicking agent. Where the modified nucleotide isincorporated close to the 5′ end of the target molecule, the 5′ terminalsingle-stranded region flanked by the nick may dissociate from thecomplementary strand, leaving behind a double-stranded target moleculeflanked with at least one 3′ single-stranded extension.

Specific nicking of the target molecule at the location(s) of themodified nucleotide(s) may be achieved by selectively cleaving one ormore of the phosphodiester bonds next to the incorporated modifiednucleotide(s) using physical, chemical or enzymatic means orcombinations of the such cleavage means.

Nature has produced a wide variety of enzymes which cumulatively respondto multiple insults on a polynucleotide molecule. Some of these insultsresult in modified nucleotides, and various enzymes are responsible fortheir repair. For example, DNA N-glycosylases excise the modifiednitrogenous heterocyclic component of the nucleotide, while APendonucleases cleave phosphodiester bonds next to the abasic (AP) sitesand DNA glycosylases/AP lyases achieve both of these functions. Incertain embodiments of the invention, enzymes that are capable ofexcising modified heterocyclic bases of modified nucleotides withspecificity have been selected in various unnatural combinations to formnicking agents for use in generating single-stranded extensions onamplified DNA fragments.

Examples of repair enzymes include:

(A) DNA N-glycosylases include the following enzymes and theirhomologues in higher eukaryotes including human homologues: Uracil DNAglycosylase (UDG) and 3-methyladenine DNA glycosylase II (AlkA)(Nakabeppu et al. J. Biol. Chem. 259:13723-13729 (1984); Varshney et al.J. Biol. Chem. 263:7776-7784 (1988); Varshney et al. Biochemistry30:4055-4061 (1991)). Additional DNA N-glycosylases include TagIglycosylase and MUG glycosylase (Sakumi, et al. J. Biol. Chem.261:15761-15766 (1986); Barret, et al. Cell 92:117-129 (1998)).

(B) AP endonucleases include Endonuclease IV of E. coli and itshomologues in higher eukaryotes including human homologue hAP1(Ljungquist J. Biol. Chem. 252:2808-2814 (1977); Levin et al. J. Biol.Chem. 263:8066-8071 (1988); Saporito et al. J. Bacteriol. 170:5141-5145(1988); Robson, et al., Nucl. Acid. Res. 19:5519-5523 (1991); Demple etal. Proc. Natl. Acad. Sci. USA 88:11450-11454 (1991); Barzilay et al.Nucl. Acid. Res. 23:1544-1550 (1995)).

E. coli Endonuclease V and homologues cleave DNA at a secondphosphodiester bond 3′ to the lesion, where the lesion may be selectedfrom any of: deoxyinosine, deoxyuridine (U), AP sites, base mismatchesas well as loops, hairpins, Flap and Pseudo-Y DNA structures (Yao, etal., J. Biol. Chem. 272:30774-30779 (1997); He et al. Mutation Research459:109-114 (2000)).

(C) DNA glycosylases/AP lyases excise selected modified nucleotides andinclude the following enzymes and their homologues in higher eukaryotesincluding human homologues:

(i) enzymes which are capable of specifically recognizing and excisingoxidized pyrimidines include E. coli Endonuclease VIII (EndoVIII), E.coli Endonuclease III (NTH) and its homologues in S. cerevisiae (NTG1and NTG2) and human (hNTH1) (Mazumder et al. Biochemistry 30:1119-1126(1991); Jiang et al. J. Biol. Chem, 272:32230-32239 (1997); Harrison etal Nucl. Acid. Res. 26:932-941 (1998); Senturker et al. Nucl. Acid. Res.26:5270-5276 (1998));

(ii) enzymes which are capable of specifically recognizing and excisingoxidized purines include E. coli FAPY-DNA glycosylase (FPG) and itshuman homologues hOGG1 and hOGG2 (Boiteux EMBO J. 6:3177-3183 (1987);Boiteux et al. J. Biol. Chem. 265:3916-3922 (1990); Tchou, et al., J.Biol. Chem. 270:11671-11677 (1995); Radicella et al. Proc. Natl. Acad.Sci. USA 94:8010-8015 (1997); Vidal et al. Nucl. Acid. Res. 29:1285-1292(2001)); and

(iii) enzymes which are specific for UV-induced cyclobutane pyrimidinedimers include T4 endo V (Gordon, et al., J. Biol. Chem. 255:12047-12050(1980); Seawell et al. J. Virol. 35:790-796 (1980)).

Certain other embodiments of the invention take advantage of thespecific functionalities of repair enzymes by creating mixtures ofcomponents that include at least one enzyme that has the desired effectof recognizing a particular modified nucleotide and excising themodified base (specificity component) and at least one enzyme thatselectively cleaves phosphodiester bond(s) adjacent to the abasicnucleotide (nicking component). The specificity of the mixtures inachieving the function of specific phosphodiester bond(s) nicking at thespecifically modified nucleotides is distinct from the functionalspecificity of individual components where the specificity component isnot able to achieve the function of the nicking component and viceversa. In general, the activity of the specificity component withrespect to the nicking component in the nicking agent should be at least2:1.

Examples of enzymes which have the specificity function, but lack theAP-site nicking function include DNA N-glycosylases. Examples of enzymeswhich have the nicking function but lack the specificity functioninclude the AP endonucleases. An artificial nicking agent may be createdby combining a DNA N-glycosylase and an AP endonuclease, for example bycombining UDG glycosylase with EndoIV endonuclease or AlkA glycosylasewith EndoIV endonuclease to achieve single-stranded cleavage at amodified nucleotide.

The choice of which components should be combined in the nicking agentto achieve single-stranded cleavage at a modified nucleotide depends on(a) the type of modified nucleotide to be excised (because thisdetermines the selection of the specificity component) and (b) the typeof strand terminus desired at the nick location after the excision ofmodified nucleotide which affects the choice of nicking component.

For example, an artificial nicking agent comprised of AlkA glycosylaseand EndoIV endonuclease has a specificity for deoxyinosine and hasnicking activity which results in a 5′ terminal deoxyribose phosphate(broken sugar) and a 3′ hydroxyl group at the nick location.

For nicking at a deoxyuridine (U) and generating a 5′ broken sugar and a3′ hydroxyl group at the nick location, an artificial nicking agent thatcontains UDG glycosylase as a specificity component and EndoIVendonuclease as a nicking component may be created.

Under certain circumstances, it may be desirable to generate a 5′phosphate at the nick location in place of the 5′ broken sugar describedabove. Accordingly, a nicking agent may be formulated with the abovedescribed specificity component but with a nicking component that leaves5′ phosphate at the nick location. Examples of nicking components withthis nicking activity include the lyase activity of DNA glycosylases/APyases, such as EndoVIII DNA glycosylase/AP lyase or FPG DNAglycosylase/AP lyase which generate 5′ phosphate and 3′ phosphate at thenick location. Consequently, the newly formulated nicking agent fornicking the target molecule at deoxyinosine might consist of acombination of AlkA glycosylase and EndoVIII glycosylase/AP lyase or FPGglycosylase/AP lyase. Alternatively, the newly formulated nicking agentfor nicking a target molecule at a deoxyuridine (U) might include acombination of UDG glycosylase and EndoVIII glycosylase/AP lyase or FPGglycosylase/AP lyase.

Under certain circumstances, it may be desirable to create a 5′phosphate and a 3′ hydroxyl group at the nick location using the abovedescribed specificity component. For example, a combination of UDGglycosylase, EndoIV endonuclease and EndoVIII glycosylase/AP lyasecreates an artificial nicking agent that specifically nicks the targetmolecule at deoxyuridine (U) generating a single nucleotide gap andleaving 5′ phosphate and 3′ hydroxyl at the nick location.

In certain embodiments of the invention, different types of modifiednucleotides may be introduced at a plurality of selected locations inorder to nick target molecule(s) sequentially at two or more locations.For example, a deoxyuridine (U), an 8-oxo-guanine, and a deoxyinosinemay be introduced into the selected locations of the target molecule(s).A single nicking agent may be formulated that includes more than onespecificity component according to the incorporated modifiednucleotides. Alternatively separate nicking agents may be formulated andapplied to the target molecule(s) sequentially. For example, AlkA andFPG glycosylase/AP lyase which selectively nicks at a deoxyinosine anddeoxy 8-oxo-guanine may be combined or used sequentially with a nickingagent that contains UDG and EndoVIII glycosylase/AP lyase thatselectively nicks at a deoxyuridine (U).

The present embodiment of the invention is further illustrated byExample II, showing preparation of two new nicking agents referred to asthe USER™ Enzyme, which specifically nicks target molecules atdeoxyuridine (U), and the USER™ Enzyme 2, which specifically nickstarget molecules at both deoxyuridine (U) and 8-oxo-guanine both leavinga 5′ phosphate at the nick location.

Applications

A prominent feature of the methods described herein is the universalityand flexibility of the approach for allowing the performance of a widerange of DNA manipulations singly or together. The universality andflexibility of these methods distinguishes them from traditionalsystems, such as restriction endonuclease-dependent cloning ormanipulation, which require laborious step-by-step experiments toperform multiple manipulations.

Examples of some uses for the present methods include:

A) Directional cloning of PCR products (exemplified in FIG. 7);

B) Site-specific mutagenesis including nucleotide or nucleotide sequencesubstitution, insertion, deletion or fusion (exemplified in FIGS. 8-12);

C) Assembly of target molecules from the plurality of intermediatefragments (exemplified in FIGS. 13-15);

D) Directional assembly of multiple target molecules into recipientmolecules (exemplified in FIG. 16);

E) Chromosomal/Enviromental DNA cloning outside the boundaries of knownsequence (chromosome walking) (exemplified in FIG. 17);

F) Construction of cDNA libraries, which may also include cloning ofcDNA beyond the boundaries of known sequences (exemplified in FIG. 18);and

G) Concurrent use of the above applications (exemplified in FIGS. 35-37.

A) Directional Cloning of PCR Products

Complementary single-stranded extensions can be generated on alinearized recipient molecule and in a PCR product, where the extensionsof each can anneal with the other to produce a recombinant moleculecapable of being introduced into competent host cells (FIG. 7).

(1) Generation of single-stranded extensions on recipient moleculesincludes construction of a recipient molecule carrying a cassette forproducing 3′ single-stranded extensions of desired length andcomposition on each ends of the linearized recipient molecule (discussedabove).

(2) Generating single-stranded extensions on a PCR fragment includesincorporation of specifically-modified nucleotides at selected positionsin the PCR fragment as described above, and nicking the fragment at themodified nucleotide with a specific nicking agent and dissociating thenicked 5′ terminal oligonucleotide from the complementary strand toproduce a fragment having single-stranded extensions of desired lengthand composition.

The primers may carry nucleotide sequences at their 5′ ends that do notanneal to the desired target. These 5′ sequences are chosen to beidentical to the single-stranded extensions on the linearized recipientmolecule, except for a terminal 3′ nucleotide, which in the primersequences is replaced by a modified nucleotide X (FIG. 7). Downstream ofthe modified nucleotide, the primer sequences are complementary to atarget molecule specific sequence to enable extension by polymerase.

Amplification of target sequence is then performed using a pair of suchprimers. After amplification, each end of the target molecule has beenextended by the additional sequence.

The resulting amplification product is then treated with a nickingagent, which specifically recognizes and nicks DNA at the locations ofmodified nucleotides. After phosphodiester bond breakage, the 5′terminal single-stranded regions beyond the nicks dissociate from thecomplementary strand, leaving the target molecule flanked by 3′single-stranded extensions. The 3′ single-stranded extensions generatedare complementary by design to the single-stranded extensions on thelinearized recipient molecule, therefore, when mixed together, thelinearized recipient molecule and target molecule assemble into arecombinant molecule (FIG. 7). Since the single-stranded extensions canbe designed to be long enough to produce stable recombinant molecules,covalent linking of DNA molecules by ligation is not always necessary.The recombinant molecule can then be introduced into competent hostcells by transformation and can be replicated.

FIG. 26 shows how unique 3′ single-stranded extensions were created on atarget molecule which was then annealed to complementary 3′single-stranded extensions on the linearized vector pNEB205A (seeExample IA). This was achieved by using a pair of primers containing adeoxyuridine as a modified nucleotide for amplification of the targetDNA and using USER™ Enzyme (see Example II) for nicking at thedeoxyuridine (U).

FIG. 26A shows primers having specific 8 nucleotide-long extensions attheir 5′ ends and which were selected to be identical to single-strandedextensions on the vector pNEB205A (FIG. 19), except for the replacementof a 3′ thymine with a deoxyuridine (U). In the presence of naturaldNTPs, Taq DNA Polymerase incorporates adenine opposite U resulting in adouble-stranded molecule which is extended on both ends by 8 base pairsand which contains primer-derived single U at each junction with thetarget molecule sequence (FIG. 26B).

In FIG. 26B, after nicking with USER™ Enzyme, the terminalsingle-stranded hepta-nucleotide dissociates from the target moleculeleaving the target molecule flanked by 3′ single-stranded extensions of8-nucleotides in length. The recipient molecule, (pNEB205A) and theUSER™ Enzyme-treated target molecule assemble into a recombinantmolecule by means of the complementary 3′ single-stranded extensions.Because of the length of the extensions, ligation is not required. Theconstruct was used to transform chemically-competent E. coli cells.

The present application is further illustrated by the Example IIIshowing the preparation of a kit for directional cloning of PCR productsby uracil excision. The efficiency of the present method is furtherillustrated by Example IV which shows that on average, 10⁵ desiredrecombinants can be obtained per 20 ng of linear vector pNEB205A, and94-95% cloning efficiency may be achieved within a wide range of PCRproduct concentration (when the host cell competency is 2×10⁷ c.f.u./μgDNA).

B) Site-Specific Mutagenesis: Nucleotide or Nucleotide SequenceSubstitution, Insertion, Deletion or Fusion

Complementary single-stranded extensions of desired length andcomposition can be generated on the ends of at least two polynucleotidemolecules. The polynucleotide molecules can then anneal via thesingle-stranded extensions to form a single target molecule (FIG. 8).This approach forms the basis for a wide variety of DNA manipulations,such as site-specific mutagenesis, deletions, insertions, gene fusionsor replacement of any DNA segment virtually at any position of thetarget molecule, and can also be applied to assembly of target moleculesfrom multiple DNA fragments with any combination of the above carriedout concurrently.

In FIG. 8, the polynucleotide molecule is identified as a targetmolecule which is amplified as two overlapping intermediate fragments inseparate amplification reactions that utilize two different sets ofamplification primers referred to as primer pair (P1/P4) and primer pair(P2/P3).

The P1 and P2 primers, although used in separate amplification reactionsfor priming the opposite strands of the target molecule, overlap eachother by a short nucleotide sequence such as 2 to 20 nucleotides. Inaddition, each overlapping primer contains one modified nucleotide whichflanks the overlap region on the 3′ side. Selection of the priming siteof P1 and P2 primers, and thereby the overlap sequence shared by the P1and P2 primers, will depend on where the desired manipulation of targetmolecule is to take place. The predetermined sequence changes thatachieve the desired manipulation are incorporated into the Primer P1 andP2 sequences.

The outside Primers P3 and P4 may prime target molecules at locationsthat enable amplification of the entire desired region of targetmolecule. The features of the outside primers are discussed in moredetail later when describing the subcloning of the assembled targetmolecules into recipient molecules.

Target molecule amplification is then performed using both sets ofprimers and when completed, both amplified fragments carry an identicalcopy of the overlap sequence, except that one fragment contains themodified nucleotide on the top strand of the overlap, while the otherfragment contains modified nucleotide on the bottom strand of theoverlap (FIG. 8 (b)).

Generation of single-stranded extensions on the amplified targetfragments may be achieved by nicking at the modified nucleotides using amodified nucleotide-specific nicking agent. The single-strandedextensions generated on the two fragments are complementary to eachother, since they are created from the overlap sequence. Thereby, theamplified intermediate fragments may be directionally assembled to yielda full-length target molecule and thereafter may be covalently linked byligation (FIG. 8 (d)). Ligation prevents the assembled target moleculesfrom dissociating. In general, ligation is necessary only if the targetmolecule is to be subjected to traditional subcloning methods, such asrestriction endonuclease-based cloning methods.

An advantage of this approach is the avoidance of side-products thatinclude the following: (a) because the single-stranded extensions do notcontain two-fold axis of symmetry, the amplified fragments cannotassemble/ligate upon themselves; (b) because of the singlenucleotide/gap, the short oligonucleotide released after modifiednucleotide excision cannot ligate back to the parental strand; and (c)because the primers used to amplify target fragments are notphosphorylated. Ligation in amplified fragments in any other orientationis not possible. Instead, a 5′ phosphate is exceptionally generated atthe location of excision of the modified nucleotide. Thus providing theonly substrate for DNA ligase.

The assembled final product, comprising the full-length target molecule,may then be subcloned into a recipient molecule of choice using, forexample, a restriction endonuclease based cloning method known in theart. For this purpose, the outside Primers P3 and P4 may be designed tocontain the unique restriction sites. After the assembly of targetfragments is accomplished, the full-length product is then subjected todigestion with restriction endonucleases, the sites for which have beenincorporated into the Primer P3 and P4 sequences, and thereafter theproduct is ligated into the recipient molecule which is pre-cleaved withthe same or compatible restriction endonucleases. An alternativeapproach to Primer P3 and P4 design is described below in (d). In thisprotocol, ligation is not used because the manipulated fragments areassembled directly into the recipient molecule without the need for arestriction endonuclease-dependent cloning step.

The general approach described above has numerous applications some ofwhich are described below.

(a) Site specific mutagenesis, including nucleotide substitution, can beachieved, for example by following the approach illustratedschematically in FIGS. 9A and 9B where the overlapping Primers P1 and P2have been specifically designed for the purpose of introducing specificnucleotide(s) changes into a target molecule.

The desired nucleotide changes may be introduced into the overlappingPrimers P1 and P2 either within the overlap sequence, as shown in FIG.9A, or downstream from the overlap sequence of either primer, as shownin FIG. 9B. During amplification, these nucleotide changes are readilyincorporated into intermediate target fragments. The resultingintermediate fragments are then treated with a nicking agent to removemodified nucleotides and to create complementary single-strandedextensions thereby permitting the subsequent assembly of targetfragments. The reconstituted full-length target molecule, however,differs from the parental target molecule, as it carries the newlyintroduced nucleotide changes. The assembled mutagenized target moleculemay then be subcloned following the cloning protocol of choice. Adetailed working example of this approach is provided in Examples V andVI and FIG. 32.

The above approach makes it possible to achieve site-specificmutagenesis in virtually any location in the target molecule, since thepriming sites may be selected anywhere along the sequence of the targetmolecule.

(b) Nucleotide sequence insertion can be achieved using the generalmethods described above. An example of how overlapping Primers P1 and P2can be designed to achieve nucleotide sequence insertion is illustratedschematically in FIG. 10. Overlapping Primers P1 and P2 may be designedfor purposes of inserting a nucleotide sequence of any desired lengthwithin the limits of oligonucleotide synthesis used to synthesize aspecific primer. The desired nucleotide sequence can be inserted intoany desired location in the target molecule.

As shown in FIG. 10, any of the nucleotide insertion sequences notpresent in the target molecule may be introduced at the 5′ ends of theoverlapping Primers P1 and P2. The overlap region may be created eitherfrom the entire additional sequence as shown in FIG. 10A or from the 5′terminal portion of the additional sequence as shown in FIG. 10B. Duringamplification, the intermediate target fragments are extended by thisadditional sequence and are flanked by the identical copy of the overlapsequence. The resulting amplification products are then treated withnicking agent to nick at the modified nucleotides and to createcomplementary single-stranded extensions thereby permitting thesubsequent assembly of intermediate target fragments into a full-lengthtarget molecule. The assembled full-length target molecule contains theinsertion sequence and can then be cloned by following the cloningprotocol of choice. This approach enables any nucleotide sequencesegment, such as a unique restriction site(s) or a polylinker sequenceto be introduced virtually at any position of the target molecule.

(c) Deletion of nucleotide sequences can be achieved using the generalmethods described above. An example of how overlapping Primers P1 and P2can be designed to achieve nucleotide sequence deletion is illustratedschematically in FIG. 11. Overlapping Primers P1 and P2 can be designedfor purposes of precisely deleting a particular nucleotide sequencesegment from the target molecule.

The overlapping Primers P1 and P2 may prime distant locations on thetarget molecule precisely adjacent to the targeted deletion region. The5′ ends of the primers must share a common overlapping sequence. Tocreate this overlap, the 5′ end of one primer is supplemented by anadditional sequence, which is a reverse-complement to the 5′ end of theother primer. During amplification, one of the two amplified fragmentsis extended by this additional sequence. Since the additional sequenceis identical to the 5′ end of the other amplified fragment, the twodistant target fragments now share the common overlap region. Theresulting intermediate target fragments are then treated with nickingagent to remove modified nucleotides and to create complementarysingle-stranded extensions thereby permitting the subsequent assembly oftarget fragments. The resulting target molecule sequence is deficient inthe precisely-deleted nucleotide sequence segment. The assembledmutagenic target molecule may then be subcloned by following the cloningprotocol of choice.

The applications in (b) and (c) above are further illustrated byExamples V and VII and FIG. 33 showing the creation of two restrictionsites and the deletion of 18-bp segment from the vector pUC19.

(d) Nucleotide sequence fusion can be achieved using the general methodsdescribed above. An example of how overlapping Primers P1 and P2 can bedesigned to achieve nucleotide sequence fusion is illustratedschematically in FIG. 12. Overlapping Primers P1 and P2 may be designedwhen desired to precisely join two distinct target molecules.

To create the common overlap region between two distinct targetmolecules, one of the overlapping primers is supplemented by anadditional sequence that is reverse-complementary to the 5′ sequence ofthe other overlapping primer. Except for the complementary overlapsequence at the 5′ end, the two primers are distinct with 3′ regionsenabling them to prime the respective targets. Optionally, theamplification reactions may be performed using two different templates(FIG. 12). During amplification, one of the two target molecules isextended at its 5′ end by the additional sequence. Since the additionalsequence is identical to the 5′ end of the other target molecule, thetwo distinct target molecules now share the common overlap region. Theresulting target molecules are then treated with nicking agent to nickat the modified nucleotides and to create complementary single-strandedextensions which can re-associate to permit assembly of the distincttarget molecules. Upon ligation, two target molecules are preciselylinked through complementary single-stranded extensions. The chimericfull-length product may then be subcloned by following the cloningprotocol of choice.

An advantage of this approach is that it provides the opportunity tocreate a fusion of two target molecules virtually at any desiredlocation without introducing undesired nucleotides into the finalconstruct. This application is further illustrated by the exampleshowing the construction of a gene fusion of E. coli Endonuclease VIIIand Mxe Intein (see Examples V and VIII and FIG. 34).

C) Assembly of Target Molecule from a Plurality of IntermediateFragments

FIGS. 13-15 present schematic illustrations of the overlapping PrimersP1 and P2 which may be designed when desired to precisely assemble atarget molecule from more than two intermediate fragments.

For example, multiple pairs of overlapping primers may be designed wherethe first pair of primers overlaps across the expected junction oftarget molecules A and B and the second pair of primers overlaps acrossthe expected junction of target molecules B and C. (FIG. 13). Theprinciples of design for individual pairs of overlapping primers aresimilar to those outlined above in FIG. 12, where one primer at its 5′end is supplemented by an overlap sequence that is reverse-complement tothe 5′ end of the other primer. Since each overlap contains a uniquesequence, the single-stranded extensions generated on individual PCRfragments may be linked only in one final combination, for example,targets A, B and C will be assembled into a combination ABC only.

An advantage of the embodiments of the invention is that the methodprovides a means to achieve multiple DNA manipulations in oneexperimental step. Each set of overlapping primers may carry thepre-selected nucleotide sequence changes necessary to perform suchmanipulations. This is further illustrated in Example IX showing theconstruction of the hincIIQ138A mutant gene which was achieved bysimultaneous construction of a gene fusion of this gene with the Mxeintein and insertion of the whole into the promoter region of the pTXB1vector. (see Examples V and IX; FIG. 35).

By designing an additional pair of primers which overlap across theexpected junction of the first and the last targets, a circular moleculemay be generated. For example, recipient molecules may be created fromthe multiple intermediate components (FIG. 14).

By designing multiple pairs of overlapping primers where each pair ofthe overlapping primers overlaps across the expected junction of theneighboring exons, the eukaryotic genes may be assembled directly fromthe genomic DNA (FIG. 15). This strategy is further illustrated by theExample showing an assembly of hAP1 gene from Human genomic DNAperformed concurrently with silent mutagenesis (see Example X; FIG. 36).

D) Directional Assembly of Multiple Target Molecules into RecipientMolecules

FIG. 16 shows a schematic illustration of how directional assembly ofmultiple target molecules into a recipient molecule can be achieved.

For example, to assemble multiple intermediate fragments, pairs ofoverlapping primers are designed according to (B) or (C) above. However,the outside primers, instead of coding for restriction sites, may bedesigned to carry at their 5′ ends, nucleotide sequences compatible witha recipient molecule such as that as described in (A) above. This allowsfor the outside ends of the assembled full-length target molecule toanneal to the single-stranded extensions on the linearized recipientmolecule, thereby creating a transformable recombinant molecule in oneexperimental step.

Advantages of this approach over the restriction endonuclease-basedcloning methodologies of the prior art include the following: (a) eachsingle-stranded extension carries unique non-palindromic sequencewhereby the amplified target fragments and linearized recipient moleculemay be directionally assembled into desired recombinant molecule; (b)the covalent linkage by ligation may not be necessary, as thesingle-stranded extensions may be made long enough to produce a stablerecombinant molecule; (c) the time-consuming procedures of restrictiondigestion, gel-purification and vector/insert ligation are omitted; and(d) multiple target molecule manipulations and cloning may be performedconcurrently in a single experimental format.

The present application is further illustrated by Example XI and FIG. 37showing the construction of 9° N_(m) V93Q Polymerase mutant bydirectional assembly of the mutagenized intermediate fragments ofpolymerase gene into a linearized pNEB205A vector.

E) Chromosomal/Environmental DNA Cloning Outside the Boundaries of KnownSequence

FIG. 17 shows a schematic illustration of a strategy for cloning DNAregions outside the boundaries of known sequences. The method is basedon generating single-stranded extensions on the amplified fragments ofunknown nucleotide sequence that are complementary to thesingle-stranded extensions on the recipient molecule. In a first step, alibrary of single-stranded flanking sequences are generated by linearamplification with one primer in the known region. To introduce apriming site at the unknown end of the amplified single-strandedflanking sequences, a homo-oligomer cytosine tail is then added at 3′termini using dCTP and terminal transferase. In the next step, thetailed single-stranded fragments are amplified using a pair of primers,which carry vector-compatible sequences at their 5′ ends. One primer,which anneals to the unknown region of the target molecule, consists ofa poly-guanine sequence and carries a modified nucleotide, for example8-oxo-guanine, at the 6^(th) position from the 5′ end. The other primer,which anneals to the known region of the target molecule, at 5′ end isalso supplemented by five guanines and an 8-oxo-guanine at the junction.

The library of the resulting double-stranded fragments are then treatedwith FPG glycosylase/AP lyase or with the nicking agent referred to asthe USER™ Enzyme 2 (see Example II) where the enzyme or agent recognizesand nicks at 8-oxo-guanine leaving each fragment in the library flankedby the identical 3′ single-stranded extensions comprised of sixcytosines. A recipient molecule carrying single-stranded extensionscomprised of six guanine residues is then used to assemble targetfragments into recombinant molecules, thus generating a library ofrecombinant molecules. Construction and preparation of linear recipientmolecules are described in Example I and FIG. 22. The library ofrecombinant molecules is then ready for transformation of competent E.coli cells.

The present application is further illustrated by Example XII showingthe cloning of an unknown genomic segment to the 3′ side of knownsequence of the super-integron from Pseudomonas alcaligenes NEB#545 (NewEngland Biolabs, Inc., Beverly, Mass.).

F) Construction of cDNA Libraries

FIG. 18 shows a schematic illustration depicting a strategy forconstruction of cDNA libraries from a total RNA sample. The first-strandsynthesis generates a library of single-stranded cDNA products fromtotal RNA using Reverse Transcriptase and oligo-dT primer containing ahexaguanine 5′ tail with an 8-oxo-guanine at the 6^(th) position fromthe 5′ end (Primer 1 in FIG. 18). A homo-oligomer cytosine tail is thenadded at 3′ termini of single-stranded cDNA products using dCTP andterminal transferase. Second-strand synthesis is then performed with DNAPolymerase I using a poly-dG primer having the 8-oxo-guanine at the6^(th) position from the 5′ end (Primer 2 in FIG. 18).

In FIG. 18, both primers contain 5′ terminal hexaguanine sequences withthe 8-oxo-guanine as the modified nucleotide close to the 5′ end.However, Primer 1 might consist of entirely poly-thymine sequence andcarry deoxyuridine, close to the 5′ end.

A library of the resulting double-stranded cDNA fragments can be treatedwith a suitable nicking agent which nicks at 8-oxo-guanine andoptionally at deoxyuridine (U) leaving each fragment in the libraryflanked by 3′ single-stranded extensions. Recipient molecules carryingthe compatible single-stranded extensions may then be used to assembletarget fragments into recombinant molecules. Examples of recipientmolecules are provided in Example IC and Example ID. The library ofrecombinant cDNA clones can then be transformed into competent hostcells.

The present invention is further illustrated by the following Examples.The Examples are provided to aid in the understanding of the inventionand are not construed to be a limitation thereof.

The references cited above and below are herein incorporated byreference.

EXAMPLE I Recipient Molecules with 3′ Single-Stranded Extensions ofDesired Length and Composition

Linearized DNA vectors having unique 3′ single-stranded extensions ofdesired length and composition were produced as shown below for vectorspNEB205A (SEQ ID NO:1), pNEB200A (SEQ ID NO:3), pNEB210A (SEQ ID NO:4)and pUC-TT (SEQ ID NO:6) (FIGS. 19-22) all of which were derived fromthe pNEB193 vector (New England Biolabs catalog 2002-2003, p. 318) byinserting a cassette into the multiple cloning site (FIG. 27A and FIG.27B). The single-stranded extensions formed in each linearized vectormolecule were designed to be non-complementary with each other to avoidproblems arising from vector termini re-annealing to form transformablecircular DNA and to provide a means to control the orientation of anytarget molecule inserted into the vector.

A) Generation of Linearized Vector pNEB205A.

A cassette 5′GCTGAGGGAAAGTCTAGATGTCTCCTCAGC (SEQ ID NO:1) containing twoinversely oriented nicking N.BbvCIB sites (CC↓TCAGC) and one XbaIrestriction site (T↓CTAGA) was inserted into the multiple cloning siteof pNEB193 plasmid. The new construct was designated as pNEB205A (FIGS.19 and 27A and B).

pNEB205A plasmid was linearized to produce 8-nucleotide 3′single-stranded extensions (FIGS. 19 and 27C) as follows: 10 μg ofpNEB205A plasmid DNA in 200 μl of NEBuffer #4 (20 mM Tris-acetate, pH7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mMdithiothreitol and 100 μg/ml bovine serum albumin) was digested with 100units of XbaI for 18 hours at 37° C. 20 units of N.BbvCIB were added tothe reaction containing XbaI-cleaved DNA and incubated for an additional1 hour at 37° C. Vector pNEB205A DNA was purified by phenol-chloroformextraction, followed by alcohol precipitation and was re-suspended in100 μl of TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).

B) Generation of Linearized Vector pNEB200A.

Cassette 5′ACGAGACTCTAGAGGATCCGTCTAGAGTCTGGT (SEQ ID NO:3) containingtwo XbaI restriction sites (T↓CTAGA) and two inversely oriented nickingN.BstNBI sites (5′-GAGCTNNNN↓, was inserted into the multiple cloningsite of pNEB193 plasmid. The new construct was designated as pNEB200A(FIG. 20).

pNEB200A plasmid was linearized to produce 8-nucleotide 3′single-stranded extensions (FIG. 20) as follows: 8 μg of pNEB200Aplasmid DNA in 200 μl of nicking buffer (10 mM Tris-HCl, pH 7.5, 10 mMMgCl₂, 150 mM KCl, 1 mM dithiothreitol) was digested with 100 units ofN.BstNBI for 1 hour at 55° C. 100 units of XbaI were added to thereaction containing N.BstNBI-nicked DNA and incubated for an additional1 hour at 37° C. Linearized vector pNEB200A DNA was purified byphenol-chloroform extraction, followed by alcohol precipitation and wasre-suspended in 50 μl of TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mMEDTA).

C) Generation of Linearized Vector pNEB210A.

Cassette 5′GCTGAGGGGGGGATCCTTTTCATTCTAGATGT CTCCTCAGC (SEQ ID NO:4)containing two inversely oriented nicking N.BbvCIB sites (CC↓TCAGC), oneBamHI restriction site (G↓GATCC) and one XbaI restriction site (T↓CTAGA)was inserted into the multiple cloning site of pNEB193 plasmid. The newconstruct was designated as pNEB210A (FIG. 21).

pNEB210A plasmid was linearized to produce 6-nucleotide 3′single-stranded extension on one end and 8-nucleotide 3′ single-strandedextension on other end of the linearized plasmid (FIG. 21) as follows:10 μg of pNEB210A plasmid DNA in 200 μl of NEBuffer #4 (20 mMTris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate,1 mM dithiothreitol and 100 μg/ml bovine serum albumin) was doublydigested with 100 units of XbaI and 10 units of BamHI for 2 hours at 37°C. 20 units of N.BbvCIB were added to the reaction containing linearizedDNA and incubated for an additional 1 hour at 37° C. Vector pNEB210A DNAwas purified by phenol-chloroform extraction, followed by alcoholprecipitation and was re-suspended in 100 μl of TE buffer (10 mMTris-HCl, pH 8.0, 0.1 mM EDTA).

D) Generation of Linearized Vector pUC-TT.

Cassette 5′ GCTGAGGGGGGGATCCTTTTCATGGATCCCCC CCTCAGC (SEQ ID NO:6)containing two inversely-oriented nicking N.BbvCIB sites (CC↓TCAGC) andtwo BamHI sites (G↓GATCC) was inserted into the multiple cloning site ofpNEB193 plasmid. The new construct was designated as pUC-TT (FIG. 22).

pUC-TT vector was linearized to produce 6-nucleotide 3′ single-strandedextensions (FIG. 22) as follows: 10 μg of pUC-TT plasmid DNA in 200 μlof NEBuffer #4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50mM potassium acetate, 1 mM dithiothreitol and 100 μg/ml bovine serumalbumin) was digested with 10 units of BamHI for 2 hours at 37° C. 20units of N.BbvCIB were added to the reaction containing linearized DNAand incubated further for 1 hour at 37° C. Vector pNEB210A DNA waspurified by phenol-chloroform extraction, followed by alcoholprecipitation and was re-suspended in 100 μl of TE buffer (10 mMTris-HCl, pH 8.0, 0.1 mM EDTA).

EXAMPLE II Preparation of Artificial Nicking Agents Specific forDeoxyuridine (U)

This Example describes the preparation of two artificial nicking agents,USER™ Enzyme and USER™ Enzyme 2 each capable of nicking adouble-stranded DNA molecule at a deoxyuridine, generating a nucleotidegap and leaving 5′ phosphate and 3′ phosphate at the nick location. Eachartificial nicking agent consists of two components. The USER™ enzymecontains UDG DNA glycosylase and EndoVIII DNA glycosylase/lyase whilethe USER™ Enzyme 2 contains UDG DNA glycosylase and FPG DNAglycosylase/lyase. One activity unit of the artificial nicking agent wasdefined as having in the mixture, sufficient amounts of the individualcomponents required to cleave to completion, 10 pmol of a 34-meroligonucleotide duplex containing a single deoxyuridine paired with adeoxyadenine in 10 μl of reaction buffer for 15 minutes at 37° C.Consequently, the optimal ratio of components in the mixture forproducing an artificial nicking agent was determined according to theunit definition.

Based on the activity unit definition of UDG glycosylase (one unit ofUDG glycosylase activity was defined as the amount of enzyme thatcatalyses the release of 60 pmol of uracil per minute fromdouble-stranded, deoxyuracil containing DNA (New England Biolabs Catalog2002-2003, p. 112)), the amount of UDG required to prepare 1 unit ofartificial nicking agent was theoretically calculated to be 0.011 unit.However, the amount of this component in the artificial nicking agentcan vary, depending on the desirability of increasing the rate ofrelease of uracil bases relative to the rate of nicking at abasic sites.Accordingly, the amount of UDG component in one activity unit of nickingagent can be increased at least 2-fold to 100-fold higher than thetheoretically requisite amounts, to a concentration of, for example0.022 to 1.0 unit of UDG.

The optimal amount of a second component, either EndoVIII or FPG,required to prepare 1 unit of the respective artificial nicking agent,USER™ Enzyme and USER™ Enzyme 2, was determined as follows:

Preparation of substrate: the double-stranded oligonucleotide substratecontaining a single deoxyuridine (FIG. 23 (SEQ ID NO:8)) was prepared asfollows: 5 μM of the 3′ and 5′ flourescein-labeled 34-meroligonucleotide containing a single deoxyuridine (U) at the 16^(th)position was mixed with 5 μM of unlabeled complementary oligonucleotidecontaining deoxyadenine at the position opposite the deoxyuridine (U) ina 1 ml of total volume and incubated for 10 minutes at 100° C. Themixture was gradually cooled down to room temperature to yield thedouble-stranded oligonucleotide.

USER™ Enzyme: Various amounts of EndoVIII protein (from 3.9 to 250 ng)were pre-mixed with 0.2 units of UDG and the resulting mixtures wereassayed for complete nicking of 10 pmol substrate in 15 min at 37° C. in10 μl reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1mM ATP, 20 μg/ml BSA). The reactions were quenched by the addition ofequal volume of 95% formamide, 0.1% xylene cyanol, 0.1% bromphenol blue,10 mM EDTA, pH 11 and the products were analyzed on a 15% TBE-Ureadenaturing gel (Invitrogen, Carlsbad, Calif.). In FIG. 24, the resultsof the activity assay showed that complete digestion of substrateoccurred with at least 31.25 ng of EndoVIII in the presence of 0.2 unitsof UDG. Accordingly to the results of this example, 1 unit of USER™Enzyme, can be prepared by mixing at least 31.25 ng of EndoVIII proteinwith 0.2 unit of UDG.

USER™ Enzyme 2: Various amounts of FPG protein (from 18.13 to 4300 ng)were pre-mixed with 0.1 unit of UDG and the resulting mixtures wereassayed for complete nicking of 10 pmol substrate for 15 min at 37° C.in 10 μl reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 10 mM DTT,1 mM ATP, 20 μg/ml BSA). The reactions were quenched by the addition ofequal volume of 95% formamide, 0.1% xylene cyanol, 0.1% bromphenol blue,10 mM EDTA, pH 11 and the products were analyzed on a 15% TBE-Ureadenaturing gel (Invitrogen, Carlsbad, Calif.). In FIG. 25, the resultsof an activity assay showed that complete digestion of substrateoccurred with at least 145 ng of FPG in the presence of 0.1 unit of UDG.Accordingly to the results of this example, 1 unit of USER™ Enzyme 2,can be prepared by mixing at least 145 ng of FPG and 0.1 unit of UDG.

EXAMPLE III Protocol and Kit for Cloning Target Molecule into pNEB205AVector

This Example describes cloning of target molecules after DNAamplification using primers containing deoxyuridine (FIGS. 26A and 26B).The amplified products are treated with the nicking agent, USER™ Enzyme(see Example II), to create unique 3′ single-stranded extensions, whichcan then anneal to the linearized vector pNEB205A carrying complementary3′ single-stranded extensions (see Example I Section A). The method isnot dependent on restriction endonuclease cleavage, nor does it requireDNA ligase for insertion of target product into vector.

A kit is provided here for use with a target molecule which has beenamplified using Taq DNA Polymerase and uracil-containing primers asspecified in FIGS. 26A and 26B.

The kit provides a cloning vector pNEB205A that has already beenlinearized and contains single-stranded extensions as described inExample I, Section A and a nicking agent, USER™ Enzyme, sufficient for10 PCR cloning reactions. The kit further provides sequencing primerssufficient for 50 sequencing reactions and an instruction manual (NewEngland Biolabs, Inc., Beverly, Mass.).

List of Components in the Kit:

Linearized Vector pNEB205A, 10 μl (0.1 μg/μl)

USER™ Enzyme, 10 μl (1 unit/μl),

Sequencing primers:

M13/pUC Sequencing Primer (−47) (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′(SEQ ID NO: 31)), 50 μl (3.2 pmol/μl),M13/pUC Reverse Sequencing Primer (−48) (5′-AGCGGATAACAATTTCACACAGGA-3′(SEQ ID NO: 32)), 50 μl (3.2 pmol/μl),Instruction manual (New England Biolabs, Inc., Beverly, MA)

The instruction manual describes how to clone the target molecule usingthe reagents provided and others that are specified. For example, themanual may contain the following advice:

(a) Amplification

Amplification of the target molecule may be achieved using Taq DNAPolymerase and a primer pair where each primer has a sequence that iscomplementary to the target molecule and additionally has a 5′ terminalsequence which is compatible with the single-stranded extension on thelinearized vector (FIG. 26A). The orientation of the target moleculewithin the vector can be reversed by exchanging the vector-specific 5′terminal sequences in between the primers.

If the target molecule is a part of a circular plasmid which carries theampicillin resistance gene, it is advisable to linearize the plasmid toavoid contamination of the PCR product with the transformable form ofthe original plasmid. Preferably, the plasmid may be digested with arestriction endonuclease that produces blunt-ended termini and/orcontains several sites in the plasmid backbone but not within the targetmolecule to be amplified.

(b) Nicking of the Amplified DNA and Assembly Reaction

The nicking agent (USER™ Enzyme) was found to be active in all PCRbuffers tested and in a variety of other commonly used buffers (TableI). The amplification product (PCR fragment) produced as discussed in(a) contains two uracil residues per molecule (FIG. 26B (b)). Oneactivity unit of USER™ Enzyme (see Example II) is capable of nicking 5pmol of the amplified DNA. If converted to mass concentration, 5 pmol ofDNA is equal to 0.33 μg for a DNA 100 bp in length or 3.29 μg for a DNA1000 bp in length.

It is preferable to insert no less than 0.011 pmol of the USER™Enzyme-treated PCR fragment into 20 ng (0.011 pmol) of the linearizedvector pNEB205A to saturate the vector. A portion of the PCR amplifiedfragments may not be compatible with the extensions on the vectormolecule because Taq DNA Polymerase may add extra bases at the 3′ end(s)of the fragments. To account for these maximum non-templated additions,insertion efficiency can be achieved using a PCR fragment concentrationof at least 3-fold higher than the vector concentration. For example,≧0.033 pmol of PCR product may be used for each 0.011 pmol (20 ng) ofvector.

Consequently, an assembly mixture may contain 10 μl PCR sample, 1 μllinearized pNEB205A (20 ng) and 1 μl USER™ Enzyme (1 unit), which isincubated for 15 minutes at 37° C. to nick the PCR products at theuracils. Complementary extensions can then be annealed to formrecombinant molecules by incubating for 15 minutes at room temperature(FIG. 26B step (e)).

T4 DNA Ligase may be added to the assembly reaction to obtaincovalently-linked recombinant molecules. This is desirable if theassembled recombinants are to be used for electroporation or otherwisefurther manipulated. If required, 1 μl of 10×T4 DNA Ligase buffer and 1μl of T4 DNA ligase may be added to the assembly reaction after 15minute incubation at 37° C. and incubated for an additional 15 minutesat room temperature.

(c) Completion of the Cloning Method

The resulting annealed recombinant molecules formed in a 2-12 μl of theassembly reaction from (b) can be used to transform chemically competentE. coli cells. Transformed cells can then be plated on either LB platescontaining 100 μg/ml ampicilin or on the LB plates containing 100 μg/mlampicilin, 0.1 mM IPTG and 0.01 mg/ml X-gal plates for the use ofblue-white selection. Using blue-white selection, transformants carryingthe recombinant molecules form white colonies, while transformantscarrying the unmodified vector form blue colonies.

The sequence of the inserted target molecule can be verified bysequencing using sequencing primers listed in the kit components above.

(d) Subcloning

If desired, the target molecule can be subcloned into another expressionvector using a variety of unique restriction sites located within theMCS of pNEB205A (FIGS. 27B and 27C). The MCS carries four unique 8-baserestriction sites (for AscI, Pad, PmeI and SbfI) flanking the insertionsite.

When a particular restriction site desired is not present in themultiple cloning site of pNEB205A, for example, such site can be readilyengineered into the target molecule by designing the PCR primers tocontain an insertion corresponding to the restriction site between thedeoxyuridine (U) and the priming sequence, as shown below:

Left primer: 5′-[GGAGACAU + (restriction site) + priming sequence]Right primer: 5′-[GGGAAAGU + (restriction site) + priming sequence].

To subclone the target molecule directly into the ATG codon of anotherexpression vector, an NdeI site can be used which is automaticallycreated within the left primer if the ATG codon is placed downstream ofthe uracil residue (5′GGAGACAUATG . . . ) (SEQ ID NO:9).

TABLE 1 USER ™ Enzyme activity in various PCR buffers and other commonlyused buffers USER ™ Reaction Buffer Buffer Composition activity (%) T4DNA Ligase 50 mM Tris-HCl pH 7.5, 10 mM (100) (NEB) MgCl₂, 10 mM DTT, 1mM ATP, 20 μg/ml BSA Thermopol Buffer 20 mM Tris-HCl pH 8.8, 10 mM KCl,100 (NEB) 10 mM (NH₄)₂SO₄, 0.1% Triton X- 100 Thermopol II 20 mMTris-HCl pH 8.8, 10 mM KCl, 100 (NEB) + 10 mM (NH₄)₂SO₄, 0.1% Triton X-4 mM MgSO₄ 100, 4 mM MgSO₄ GeneAmp Buffer 10 mM Tris-HCl pH 8.3, 50 mMKCl, 200 (Applied 1.5 mM MgCl₂, 0.001% (w/v) gelatin Biosystems) GeneAmpBuffer II 10 mM Tris-HCl pH 8.3, 50 mM KCl, 100 (Applied 4 mM MgCl₂Biosystems) + 4 mM MgCl₂ PCR buffer 10 mM Tris-HCl pH 8.3, 50 mM KCl,100 (Roche) 1.5 mM MgCl₂ Taq Pol Buffer 10 mM Tris-HCl pH 9.0, 50 mMKCl,  75 (Promega) 0.1% Triton-X-100, 2.5 mM MgCl₂, PCR Buffer pH 8.7,1.5 mM MgCl₂, 100 (Qiagen) Additional components unknown TE Buffer 10 mMTris-HCl pH 8.0, 0.1 mM 100 EDTA, TE Buffer + 10 mM Tris-HCl pH 8.0, 0.1mM 100 2 mM MgSO₄ EDTA, 2 mM MgSO₄ TE Buffer + 10 mM Tris-HCl pH 8.0,0.1 mM 100 4 mM MgSO₄ EDTA, 4 mM MgSO₄ TE Buffer + 10 mM Tris-HCl pH8.0, 0.1 mM  75 6 mM MgSO₄ EDTA, 6 mM MgSO₄ (NEB—New England Biolabs,Inc., Beverly, MA) (Applied Biosystems, Foster City, CA) (RocheDiagnostics GmbH, Mannheim, Germany) (Qiagen, Studio City, CA)

EXAMPLE IV Cloning of the Chloramphenicol Resistance Gene (Cat) intoVector pNEB205

Using the methodology described in Example III, the cat (Cm^(r)) genewas amplified as a 950 bp fragment of the pGPS2.1 plasmid with Taq DNAPolymerase and primers listed below:

Left primer: (SEQ ID NO: 33) 5′-GGAGACAUCGGATCCATACCTGTGACGGAAGRight primer: (SEQ ID NO: 34) 5′-GGGAAAGUGGATCCAGGCGTTTAAGGGCACC

The 50 μl PCR reactions contained 5 μl of 10× GenAmp PCR buffer (AppliedBiosystems, Foster City, Calif.), 20 ng of the pGPS2.1 (New EnglandBiolabs, Inc., Beverly, Mass.) DNA, 200 μM dNTPs, 0.2 μM of each primerand 2 units of Taq DNA Polymerase. The cat gene was amplified for 8, 9,10, 11, 13, 16, 20, 25 and 30 cycles using the cycling protocol below:

94° C.  5 min 94° C. 30 sec 55° C.  1 min one cycle 72° C. 40 sec 72° C. 5 min

The amount of PCR product in a 10 μl sample (20% of the total PCRvolume) was evaluated by gel electrophoresis (FIG. 28) and was estimatedto be 5, 10, 17, 45, 82, 164, 215, 346 or 390 ng, respectively.

10 μl of each PCR sample was mixed with 1 μl (20 ng) of linearizedvector pNEB205 and 1 μl (1 unit) of USER™ Enzyme. Reactions wereincubated for 15 minutes at 37° C. to cleave at deoxyuracil residues andan additional 15 minutes at room temperature to allow annealing of thecomplementary extensions.

50 μl of chemically competent E. coli ER2267 cells (cell competency2×10⁷ c.f.u./μg pNEB205A) were transformed with 3 μl of the assemblyreaction. Transformants were selected by plating 3×25 μl of thetransformation reaction (from 1.5 ml total transformation reactionvolume) on LB plates supplemented with Amp+IPTG+X-gal. The white(recombinant) and blue (vector background) colonies were counted after18 hours at 37° C. Cloning efficiency was determined by calculating thefraction of white colonies compared to the total number oftransformants. The cat gene cloning into pNEB205A results are shown inFIG. 29 and FIG. 30.

EXAMPLE V Protocols and Kit for Target Molecule Manipulation

This Example describes how various DNA manipulations may be performedsuch as site-specific mutagenesis, generation of target moleculefusions, deletion, insertion or replacement of any DNA segment virtuallyat any position of the target molecule, assembly of target moleculesfrom multiple DNA fragments and any combination of the aboveapplications simultaneously (FIGS. 8-15) using deoxyuridine as themodified nucleotide (X).

Target molecule(s) are amplified by generating multiple overlappingfragments. To create the overlap region between two neighboringfragments of the target molecule, the amplification primers are designedto have an overlapping sequence at their 5′ ends with one deoxyuridineflanking the overlap sequence on the 3′ side (FIG. 31).

The primer design shown in FIG. 31 can be further modified toincorporate sequence changes at or near the overlap region to producethe specific desired DNA manipulation (FIGS. 9-15). After amplification,the sequence changes become incorporated into the amplified PCRfragments. The adjoining fragments are flanked by a common overlapregion, but one fragment contains a deoxyuridine (U) on the top strandof the overlap region, while the other fragment contains a deoxyuridine(U) on the bottom strand of the overlap region. Single-strandedextensions can then be created at the junction of neighboring fragmentsby excising deoxyuridine (U) residues with the USER™ Enzyme. Thesingle-stranded extensions, generated from fragments designed to adjoin,are complementary to each other, as they consist of the overlapsequence. The fragments can be directionally assembled to yield afull-length target molecule with sequence changes.

The particulars of this Example are abstract protocols to explain howany DNA can be modified in a predetermined manner (FIGS. 32-37) using akit containing reagents and the appropriate protocol of the typeprovided by (a)-(d) below.

(a) Design of Primers

A target molecule can be manipulated by using multiple primer pairs asshown in FIG. 31. Overlapping Primers P1 and P2 may overlap each otherby 2-20 nucleotides on their 5′ ends while priming opposite DNA strandson the target molecule. The priming site can be selected on the targetmolecule sequence at or near the point of manipulation. In both of theoverlapping primers, the 3′-nucleotide flanking the overlap sequence canbe deoxyuridine (U). Preferably, the nucleotide sequence within theoverlap does not contain a two-fold axis of symmetry, thus avoidingself-complementarity. Where the sequence of target DNA at the point ofmanipulation does not display a suitable priming sequence for theoverlap, a sequence change can be introduced to provide a necessary A orT, for example, at the degenerate third position of a codon.

The sequences of Primers P3 and P4 may complement sequences on thetarget molecule containing unique restriction sites, which can then beused for subsequent cloning of the assembled target molecule.Alternatively, a unique restriction site might be introduced into the 5′end of the P3 and P4 primer sequence.

(b) Amplification

A target molecule can be amplified with Taq DNA Polymerase as twooverlapping fragments in separate PCR reactions using (P1+P3) and(P2+P4) primer pairs. The result of amplification is two fragments inwhich one terminus in each occurs at the position of interest (the“split site”). At the split site, each PCR fragment is flanked by anidentical copy of the overlap, except that uracils are positioned onopposite DNA strands (FIG. 8). The PCR fragments should preferably bepurified and adjusted to a concentration of 0.1-1.0 μM.

Because Taq DNA Polymerase introduces non-template nucleotides at the 3′termini of amplified PCR fragments, end polishing of PCR fragments issuggested for achieving accurate and efficient DNA manipulation. Endpolishing can be achieved as follows:

For a 50 μl reaction, no more than 10 pmol of PCR Fragment is mixed with5 μl 10×T4 DNA ligase buffer, 1 μl 10 mM dNTP, 1 μl DNA Polymerase ILarge (Klenow) Fragment. H₂O is added to a final volume of 50 μl. Themixture is incubated for 10 minutes at 37° C. to remove non-template 3′nucleotides followed by a further incubation for 20 minutes at 80° C. toinactivate the Klenow Fragment.

(c) Generation of 3′ Single-Stranded Extensions Using a Nicking Agent

3′ single-stranded extensions can be generated on the polished PCRfragments with the USER™ Enzyme by nicking the PCR fragments on the 3′and 5′ sides of the deoxyuridine (U) and dissociation of the shortoligonucleotide created by the nick to form fragments flanked by a 5′phosphate on one strand and a 3′ single-stranded extension on the otherstrand. The nicking is achieved by supplementing the 50 μl reaction from(b) with 1 unit (1 μl) of the USER™ Enzyme (see Example II) andincubating for 15 minutes at 37° C.

If the PCR fragment contains two uracils per molecule, one activity unitof the USER™ Enzyme is capable of nicking no more than 5 pmol of PCRproduct under the reaction conditions defined above.

(d) Assembly Reaction

The preparations of individual PCR fragments containing complementarysingle-stranded extensions described in (c) can then be combined inequimolar amounts. 1 μl T4 DNA Ligase is added to the mixture of PCRfragments and the mixture is incubated for 30 minutes at roomtemperature to ligate the PCR fragments. 1/20 volume of the ligationreaction is then retrieved and run on an agarose gel to check forligation efficiency. If the ligation yield is found to be satisfactory,T4 DNA Ligase is inactivated by heating for 20 minutes at 80° C.

Use of equimolar amount of PCR fragments provides a maximum yield ofligated fragments and is especially important when ligating more thantwo PCR fragments, otherwise the under-represented fragment will limitthe final product formation. However, in some cases, due to thevariations in the nucleotide composition and/or length ofsingle-stranded extensions and PCR fragment sizes, the concentrationratio that produces the highest yield of the final ligation product maybe optimized in a pilot ligation prior to a large-scale ligationreaction.

(e) Cloning of Ligated Product into the Vector of Choice

The ligated product can be digested with appropriate restrictionendonucleases that recognize sequences engineered into the outsidePrimers P3 and P4 and cloned using traditional protocols which includegel-purification and ligation of the target fragment into the vector ofchoice that has been pre-cleaved with the same or a compatiblerestriction endonuclease. Alternatively, the ligated product can beintroduced into a vector according to Examples III and IV.

In the present Example, a kit is provided which contains reagents andprotocols appropriate for use with the PCR amplified DNA fragments whichhave been manipulated as specified in FIGS. 8-15. The kit describedbelow contains sufficient reagents for 10 reactions.

List of Components in the Kit:

-   -   10×T4 DNA Ligase Buffer, 500 μl;    -   10 mM dNTP solution, 100 μl;    -   DNA Polymerase I, Large (Klenow) Fragment, 20 μl (5 units/μl);    -   USER™ Enzyme, 10 μl (1 unit/μl)    -   T4 DNA Ligase, 20 μl (400 units/μl)    -   Instruction manual

For convenience, all enzymes in the kit have been adjusted to perform inthe T4 DNA Ligase buffer to avoid buffer changes during the reaction.

EXAMPLE VI Site-Specific Mutagenesis of HincII Restriction Endonuclease

The design of two primer pairs P1/P4 and P2/P3 and their use inmodifying hincIIR endonuclease gene is shown in FIG. 32. Primer P1 codedfor a substitution of codon CAA with the codon TTT. 420 bp and 380 bpfragments of the hincIIR gene were amplified using Taq DNA Polymeraseand primer pairs P1/P4 and P2/P3, respectively. The total volume foreach PCR reaction was 6 tubes of 100 μl. After amplification, the PCRproducts of six identical reactions were combined, purified byphenol-chloroform extraction and alcohol precipitation and dissolved in50 μl of TE buffer. DNA concentration was determined by gelelectrophoresis and was estimated to be 0.2 mg/ml for each PCR fragment.Two 50 μl reactions were set up as follows:

-   -   25 μl (˜20 pmoles) of either 380 bp or 420 bp PCR fragment    -   5 μl of 10×T4 DNA Ligase buffer    -   1 μl of 10 mM dNTPs    -   18 μl of Milli-Q™ H₂O    -   1 μl (5 units) of Klenow Fragment

The reactions were incubated for 10 minutes at 37° C. to formblunt-ended PCR fragments. Klenow Fragment was inactivated by incubatingfor 20 minutes at 80° C. 1 μl (1 unit) of USER™ Enzyme was added to eachreaction and samples were incubated for 15 minutes at 37° C. Afterincubation, the reactions were placed on ice.

Pilot ligation: 1 μl of each PCR fragment was combined with 7 μl of 1×T4 DNA Ligase buffer. 1 μl (40 units) of T4 DNA Ligase was added andincubated for 30 minutes at room temperature. The ligation reaction wasanalyzed by gel electrophoresis in parallel with 1 μl of eachUSER™-treated fragment to evaluate the ligation efficiency (FIG. 32(e)).

Large-scale ligation: 20 μl of each PCR fragment were combined, 1 μl(400 units) of T4 DNA Ligase was added and incubated for 30 minutes atroom temperature. A 2 μl aliquot was assayed by gel eletrophoresis toevaluate ligation efficiency. T4 DNA Ligase was inactivated byincubating for 20 minutes at 80° C. The ligation mixture was digestedwith NdeI and SapI restriction endonucleases. A 780 bp NdeI-SapIfragment carrying the complete hincIIR/Q138F gene was purified from theagarose gel and cloned into NdeI-SapI cleaved dephosphorylated pTXB1vector (New England Biolabs, Inc., Beverly, Mass.).

EXAMPLE VII Insertion of Unique Restriction Sites and Deletion of 18 bpSegment from pUC19 Vector DNA

The design of two primer pairs P1/P4 and P2/P3 and their use ininserting restriction sites and deleting a sequence from pUC19 isillustrated in FIG. 33. The priming sites for the Primers P1 and P2 onpUC19 were separated by the 18 base pair sequence that was to bedeleted. To create the 6 bp-long overlap region, Primers P1 and P2 weresupplemented by six-nucleotide insertion sequences at their 5′ ends,which also created BsrGI and AvrII restriction sites. Two fragments ofpUC19 were amplified (one of 610 bp and one of 810 bp) with Taq DNAPolymerase and primer pairs P1/P4 and P2/P3, respectively. Six identical100 μl PCR reactions were carried out. After amplification, the PCRproducts combined (each fragment separately), were purified byphenol-chloroform extraction and alcohol precipitation and dissolved in50 μl of TE buffer. The 610 bp PCR fragment concentration was 0.15 mg/ml(0.38 pmol/μl); the 810 bp fragment concentration was 0.3 mg/ml (0.57pmol/μl). An end-polishing reaction (100 μl) was set up as follows:

62 μl of Milli-Q™ H₂O

15 μl (5.7 pmoles) of 610 bp fragment

10 μl (5.7 pmoles) of 810 bp fragment

10 μl of 10×T4 DNA Ligase buffer

2.0 μl of 10 mM dNTPs

1.0 μl (5 units) of Klenow Fragment

The reaction mixture was incubated for 10 minutes at 37° C. to polishends of the PCR fragments. Klenow Fragment was then inactivated byincubating for 20 minutes at 80° C. 1 μl of the USER™ Enzyme was thenadded to the reaction mixture and incubated for 15 minutes at 37° C. 1μl of T4 DNA Ligase was then added and incubated for 30 minutes at roomtemperature. T4 DNA Ligase was inactivated by incubating for 20 minutesat 80° C. A 10 μl aliquot from the ligation mixture was digested eitherwith BsrGI or AvrII and assayed by gel electrophoresis to check for thepresence of these newly-introduced restriction sites within the 1420 bpligation product (see FIG. 35B). The 1420 bp ligation product, nowcarrying unique BsrGI and AvrII sites, was cleaved with BsaI andHindIII; the 1380 bp DNA fragment was purified from the agarose gel andsubcloned into BsaI and HindIII-cleaved dephosphorylated pUC19.

EXAMPLE VIII Construction of E. coli Endonuclease VIII Mxe Intein GeneFusion

The design of two primer pairs P1/P4 and P2/P3 and their use inconstructing an endonuclease gene is shown in FIG. 34. Seven-nucleotidelong overlap sequences for P1 and P2 primers consisted of the last twonucleotides of the EndoVIII gene and the first five nucleotides of theMxe Intein gene. An 800 bp EndoVIII gene was amplified from E. coligenomic DNA using Taq DNA Polymerase and primer pair P2/P3. A 265 bp 5′terminal fragment of the Mxe Intein gene was amplified from pTXB1 usingTaq DNA Polymerase and primer pair P1/P4. The total amplificationreaction volume for each PCR was 6 tubes of 100 μl.

After amplification, the PCR products were purified using QIAquick™ PCRPurification Kit (Qiagen, Studio City, Calif.). PCR fragmentconcentration was calculated from gel electrophoresis to be 0.1 mg/ml(0.2 pmol/μl) for the 800 bp PCR fragment; and 0.05 mg/ml (0.3 pmol/μl)for the 265 bp PCR fragment. The 40 μl reaction was set up as follows:

4 μl of Milli-Q™ H₂O

10 μl (2 pmoles) of 800 bp fragment

20 μl (6 pmoles) of 265 bp fragment

4 μl of 10×T4 DNA Ligase buffer

1.0 μl of 10 mM dNTPs

1 μl (5 units) of Klenow Fragment

The PCR fragment concentration ratio 1:3 was chosen to avoidcontamination of the 1065 bp ligation product with the initial 800 bpPCR fragment. The reaction mixture was incubated for 10 minutes at 37°C. to polish the ends of PCR fragments. Klenow Fragment was inactivatedby incubating for 20 minutes at 80° C. 1 μl of the USER™ Enzyme wasadded to the reaction mixture and incubated for 15 minutes at 37° C. 1μl of T4 DNA Ligase was added and ligation proceeded for 30 minutes atroom temperature. A 2 μl aliquot of the ligation mixture was assayed bygel electrophoresis (see FIG. 36B). T4 DNA Ligase was inactivated byincubating for 20 minutes at 80° C.

The 1065 bp ligation product comprised of the EndoVIII gene fused to the5′-terminus of the Mxe Intein gene, was cleaved with NdeI and AatII,gel-purified and subcloned into NdeI- and AatII-cleaved dephosphorylatedpTXB1 vector.

EXAMPLE IX Construction of the HincIIR/Q138A Mutant and its ConcurrentInsertion into pTXB1 Vector Between the Promoter Region and Mxe InteinGene

The experimental scheme for construction of a mutant gene and insertionof the mutant gene into a vector is shown in FIG. 35. FIG. 35 (a) showsa schematic illustration of the desired product, which was the productof the present Example. Four PCR primer pairs P1/P2, P3/P4, P5/P6 andP7/P8 were designed as depicted in FIG. 35 (b) including three sets ofthe overlapping primers, P2/P3, P4/P5, P6/P7 respectively.

The overlapping Primers P2 and P3 created the fusion between thepromoter region of pTXB1 vector and the 5′ terminus of the hincIIR gene.An 11-nucleotide long overlap sequence included four nucleotides ofpTXB1 sequence and the first seven 5′ terminal nucleotides of thehincIIR gene.

The overlapping Primers P4 and P5 introduced the codon substitution atthe position 138 of the hincIIR gene. In Primer P5, wild type codon CAA(coding for a glutamine) was replaced by a mutagenic codon GCT, whichcoded for an alanine. In Primer P4, correspondingly, the sequence TTGwas replaced by the sequence AGC.

The last set of the overlapping primers, P6 and P7, created a fusionbetween the 3′ terminus of the hincIIR gene and a 5′ terminus of the MxeIntein gene. A 9-nucleotide long overlap sequence included the last four3′ terminal nucleotides of the hincIIR gene and first five nucleotidesof the Mxe Intein gene. Primers P1 and P8 both primed pTXB1 vectorsequence across the unique XbaI and BsrGI sites.

Four separate PCR reactions were performed using Taq DNA Polymerase andthe respective primer pair. A 140 bp promoter region of pTXB1 vector wasamplified using primer pair P1/P2 (PCR1) and pTXB1 plasmid as atemplate. The 420 bp and 380 bp fragments of the hincIIR gene wereamplified using a full-length hincIIR gene as a template and either theprimer pair P3/P4 (PCR2) or primer pair P5/P6 (PCR3), respectively. A360 bp 5′-terminal fragment of Mxe Intein gene was amplified from pTXB1vector using primer pair P7/P8 (PCR4). After amplification, each PCRfragment was purified by phenol-chloroform extraction and alcoholprecipitation and dissolved in 50 μl of Milli-Q™ H₂O. The PCR fragmentconcentration was calculated from the OD₂₆₀ measurements: PCR1: 0.23mg/ml (2.9 pmol/μl); PCR2: 0.35 mg/ml (1.3 pmol/μl); PCR3: 0.30 mg/ml(1.2 pmol/μl) and PCR4: 0.30 mg/ml (1.3 pmol/μl).

Four 50 μl reactions were set up as follows:

5 μl of 10×T4 DNA Ligase buffer

1.0 μl of 10 mM dNTPs

1 μl (5 units) of Klenow Fragment

28 μl of Milli-Q™ H₂O

15 μl (44 pmoles) of PCR1

(or 15 μl (20 pmoles) of PCR2)

(or 15 μl (18 pmoles) of PCR3)

(or 15 μl (20 pmoles) of PCR4)

Each reaction mixture was incubated for 10 minutes at 37° C. to polishthe ends of PCR fragments. Klenow Fragment was inactivated by incubatingfor 20 minutes at 80° C. Since 420 bp and 380 bp hincIIR fragmentscontain two uracils per molecule, 2 units of USER™ Enzyme are necessaryto completely nick 20 pmol of each fragment. Therefore, 2 μl of theUSER™ Enzyme was added to each reaction mixture and incubated for 15minutes at 37° C. After incubation, the reactions were placed on ice.

Pilot ligation (FIG. 35 step (f)): 1 μl of each PCR fragment wascombined in 5 μl of 1×T4 DNA Ligase buffer. 1 μl of T4 DNA Ligase wasthen added and PCR fragments were ligated for 30 minutes at roomtemperature. The ligation reaction was analyzed by gel-electrophoresisin parallel with 1 μl of each USER™-treated fragment to evaluateligation efficiency.

Large-scale ligation: 20 μl of PCR1, PCR2 and PCR3 fragment and 40 μl ofPCR4 fragment were combined together, 1 μl of T4 DNA Ligase was addedand ligation proceeded for 30 minutes at room temperature. In thelarge-scale ligation, the concentration of PCR4 fragment was doubled asthe pilot ligation showed the significant accumulation of the 900-bppartial ligation product. A 5 μl aliquot from the ligation mixture wasassayed by gel electrophoresis. T4 DNA Ligase was inactivated byincubating for 20 minutes at 80° C. The 1270 bp ligation product carriedthe hincIIR/Q138A gene precisely inserted between the promoter region ofpTXB1 and the 5′ terminus of the Mxe Intein gene. It was then cleavedwith XbaI and BsrGI restriction endonucleases, purified from the agarosegel and subcloned into XbaI and BsrGI cleaved, dephosphorylated pTXB1vector.

EXAMPLE X Assembly of hAP1 Gene from Human Genomic DNA in Combinationwith Silent Mutagenesis

The experimental scheme for assembly of the hAP1 gene in combinationwith silent mutagenesis is shown in FIG. 36. FIG. 36 (a) shows aschematic illustration of the cDNA of the human AP1 endonuclease gene.Five PCR primer pairs P1/P2, P3/P4, P5/P6 P7/P8 and P9/P10 were designedas depicted in FIG. 36 (b) including four sets of the overlappingprimers, P2/P3, P4/P5, P6/P7, P8/P9, respectively.

The overlapping Primers P2 and P3 created the junction between the Exon2 and Exon 3 of hAP1 gene. An 8-nucleotide long overlap region wasselected within the 5′ terminal sequence of Exon 3. Primer P2 annealedto the 3′ terminal region of Exon 2, but at the 5′ end it wassupplemented with a 20 nucleotide-long sequence that was the reversecomplement of the 5′ terminal sequence of Exon 3.

The overlapping Primers P4 and P5 created the junction between Exon 3and Exon 4 of hAP1 gene. A 9-nucleotide overlap region was formed fromthe last four nucleotides of Exon 3 and five first nucleotides of Exon5.

The overlapping Primers P6 and P7 created the junction between Exon 4and Exon 5 of hAP1 gene. A 9-nucleotide overlap region was formed fromthe last four nucleotides of Exon 4 and the five first nucleotides ofExon 5.

The overlapping Primers P8 and P9 introduced a silent substitutionwithin the Exon 5 sequence in order to eliminate the naturally occurringNdeI restriction site. Within the 9 nucleotide overlap region Primer P8carried a T to C substitution and Primer P9 carried an A to Gsubstitution such that the NdeI site CATATG was converted into CGTATC.

The NdeI restriction site was then engineered into the 5′ sequence ofthe Primer P1 to create a restriction endonuclease site for subcloningof the assembled hAP1 gene. For the same purpose, a SapI site wasengineered into the 5′ sequence of the Primer 10.

Five separate PCR reactions were performed using Taq DNA Polymerase andtotal human genomic DNA as a template. An 80 bp Exon 2 was amplifiedusing primer pair P1/P2 to yield a product referred to as PCR1. A 180 bpExon 3 was amplified using primer pair P3/P4 yielding product referredto as PCR2. A 200 bp Exon 4 was amplified using primer pair P5/P6yielding product referred to as PCR3. An 80 bp 5′ terminal portion ofExon 5 was amplified using primer pair P7/P8 yielding product referredto as PCR4 and a 460 bp 3′ terminal portion of Exon 5 was amplifiedusing primer pair P9/P10 yielding product referred to as PCR5. Afteramplification, each PCR fragment was purified by phenol-chloroformextraction and alcohol precipitation and dissolved in 50 μl of Milli-Q™H₂O. PCR fragment concentration was measured to be: PCR1: 0.005 mg/ml(0.10 pmol/μl); PCR2: 0.03 mg/ml (0.26 pmol/μl); PCR3: 0.10 mg/ml (0.77pmol/μl); PCR4: 0.01 mg/ml (0.20 pmol/μl); PCR5: 0.05 mg/ml (0.17pmol/μl).

Five 50 μl reactions were set up as follows:

5 μl 10×T4 DNA Ligase buffer

1.0 μl 10 mM dNTPs

1 μl Klenow Fragment

40 μl (4 pmol) of PCR1 or PCR2 or PCR3 or PCR4 or PCR5

Milli-Q™ H₂O up to 50 μl.

Each reaction mixture was incubated for 10 minutes at 37° C. to formblunt ended PCR fragments. Klenow Fragment was inactivated by incubatingfor 20 minutes at 80° C. 2 μl (2 units) of USER™ Enzyme was added toeach reaction mixture and incubated for 15 minutes at 37° C. Afterincubation, the reactions were placed on ice.

Pilot ligation (FIG. 36 stein (f)): PCR fragments were combined in thefollowing ratio (μl):

PCR1:PCR2:PCR3:PCR4:PCR5=4:1:1:1:3

1 μl of T4 DNA Ligase was added and ligation proceeded for 30 minutes atroom temperature. The ligation reaction was analyzed by gelelectrophoresis in parallel with 1 μl of each PCR fragment to evaluateligation efficiency.

Large-scale ligation: 10-fold higher amounts of PCR fragments werecombined together in the same ratio as shown in the pilot ligation, 1 μlof T4 DNA Ligase was added and ligation proceeded for 30 minutes at roomtemperature. 5 μl aliquot from the ligation mixture was assayed by gelelectrophoresis. T4 DNA Ligase was inactivated by incubating for 20minutes at 80° C.

The 1000 bp ligation product carried the assembled hAP1 gene with theinactivated NdeI restriction site at position 490 nt. The ligatedfragment was then cleaved with NdeI and SapI, purified from the agarosegel and subcloned into NdeI and SapI cleaved dephosphorylated pTXB1vector.

EXAMPLE XI Site-Specific Mutagenesis of 9° N_(m) Polymerase byDirectional Assembly of the Mutagenized PCR Fragments into LinearizedpNEB205A Vector

A summary of the experimental approach used here to achievesite-specific mutagenesis is shown in FIG. 37. Two PCR primer pairs weredesigned as depicted in FIG. 37. Overlapping Primers P1 and P2 coded forthe codon 93 change from GTC (coding for Val93) to CAA (coding forGln93). Primer P1 carried two nucleotide changes: TC substitution withthe AA. Primer 2 carried three nucleotide changes: GAC substitution withthe TUG. Primers P3 and P4 at their 5′ ends were supplemented with anadditional 8-nucleotides that were compatible with the single-strandedextensions on linearized pNEB205A vector (see Example II). 2041 bp and287 bp fragments of the 9° N_(m) Polymerase gene were amplified usingTaq DNA Polymerase and primer pairs P1/P4 and P2/P3, respectively. Thetotal reaction volume for each PCR was 100 μl. The assembly reaction wasset-up as follows:

1 μl (0.1 pmol) of 287 bp PCR fragment

10 μl (0.1 pmol) of 2041 bp PCR fragment

1 μl pNEB205A linear vector (20 ng)

1 μl USER™ Enzyme (1 unit)

The reaction was incubated for 15 minutes at 37° C., and then for 15minutes at room temperature. 50 μl of E. coli ER2267 competent cellswere transformed with 5 μl of the above reaction. 100 μl of thetransformation reaction (out of total 1 ml volume) was plated on LBplates containing 100 μg/ml ampicilin.

More than 2×10³ transformants were recovered after 18 hours incubationat 37° C. Out of these, plasmid DNA was purified from 6 individualtransformants and assayed for the presence of insert by cleavage withrestriction endonuclease BbvCI. All of them carried the 2.3 kb insert,indicating that full-length 9° N_(m) Polymerase gene was cloned. Five ofsix plasmids were sequenced across the mutagenized region and found thatall of them carry desired codon substitution: GTC to CAA.

EXAMPLE XII Cloning of the 3′ Genomic Region of the Super-Integron fromPseudomonas alcaligenes NEB#545

An outline of the strategy used to clone the unknown 3′ region of thesuper integron from Pseudomonas alcaligenes NEB#545 (New EnglandBiolabs, Inc., Beverly, Mass.) downstream of the 3′ end of the contig C(Vaisvila et al. Mol. Microbiol. 42:587-601 (2001)) is shown in FIG. 38.

Three PCR primers were designed. Primer Pal3-1(GGAACGGCAATTGGCCTTGCCGTGTA (SEQ ID NO:28) was used for linearamplification of the 3′ genomic DNA segments.

Primer Pal3-3 (GGGGGXCTAAAGCCTGCCCCTTAACCAAAC GTTA (SEQ ID NO:29), whereX is 8-oxo-Guanine) and Primer GG-2 (GGGGGXGGGGGGGGGGGGHN (SEQ IDNO:30), where H is either A, T, or C; N is either A, T, C, or G; and Xis 8-oxo-Guanine) were used for nested PCR amplification.

A library of single-stranded fragments downstream of the known 3′ end ofthe contig C was generated from the total genomic DNA by linearamplification using the protocol below. 100 ng of Pseudomonasalcaligenes genomic DNA in a 100 μl of ThermoPol reaction buffercontaining 10 pmol Pal3-1 primer, 200 μM dNTP and 2.5 units of Taq DNAPolymerase was linearly amplified for 25 cycles using the cyclingprotocol below:

94° C.  4 min 94° C. 30 sec 57° C.  1 min one cycle 72° C.  1 min

After amplification, the resulting amplification products were purifiedon Microcon-PCR Filter Unit (Millipore Corporation, Bedford, Mass.).

For the cytosine tailing reaction, 10 μl of the purified amplificationproducts were incubated with 20 units of Terminal Transferase for 15minutes at 37° C. in a NEBuffer 4 reaction buffer containing 0.25 mMCoCl₂ and 2 mM dCTP. Terminal Transferase was then inactivated byincubating at 75° C. for 10 minutes.

To create a library of the double-stranded products, 2 μl of the tailedamplification products were further amplified for 25 cycles with Pal3-3and GG-2 primers in a 100 μl of ThermoPol reaction buffer containing 10pmol of each primer, 200 μM dNTP and 2.5 units of Taq DNA Polymeraseusing the cycling protocol below:

94° C.  4 min 94° C. 30 sec 57° C.  1 min one cycle 72° C.  1 min 72° C. 5 min

To introduce the amplified library of PCR products into pUC-TT, a 9 μlof PCR sample were combined with 1 μl of the linearized pUC-TT vectorand 1 μl (8 units) of the FPG glycosylase and incubated for 15 minutesat 37° C. to cleave at 8-oxo-guanine residues. The reaction wasincubated for additional 15 minutes at room temperature to allowannealing of the complementary extensions.

Chemically-competent E. coli ER1992 cells were transformed with 10 μl ofthe assembly reaction. Recombinants were selected by plating thetransformation reaction (LB+Amp) plates. More than 4×10³ transformantswere recovered after 18 hours incubation at 37° C. 27 out of total 32individual transformants that were screened by colony PCR carried theinsert varying from 0.3 kb to 1.3 kb in length. Plasmid DNA from fiveindividual transformants that carried the longest inserts (from 0.9 kbto 1.3 kb) was purified and sequenced across the insert region. All ofthem carried the same genomic DNA segment that is localized on the 3′side from the known region of the Pseudomonas alcaligenes NEB#545super-integron.

What is claimed is:
 1. A method of generating a single-strand region ona polynucleotide molecule comprising: (a) providing in a reactionmixture, a polynucleotide molecule having a single modified nucleotideon one strand of a duplex at a specific location; and (b) enzymaticallycleaving the polynucleotide molecule at the modified nucleotide, byadding two or more enzymes to a reaction mixture wherein at least one ofthe enzymes is a DNA glycosylase and at least one of the enzymes is acleaving enzyme; and wherein the DNA glycosylase and the cleaving enzymehaving an activity ratio of at least 2:1 in the reaction mixture, theactivity ratio being the ratio of a first rate of forming an abasic siteby excision of the modified nucleotide from the polynucleotide by DNAglycosylase to a second rate of cleaving at the abasic site in thepolynucleotide by the cleaving enzyme, the first and second ratesdetermined for a single predetermined molar amount of the modifiednucleotide in the polynucleotide.
 2. A method according to claim 1,wherein the polynucleotide molecule is a product of a primerpair-dependent DNA amplification of a target molecule.
 3. A methodaccording to claim 2, wherein introducing the modified nucleotide intothe polynucleotide further comprises introducing the modified nucleotideinto one or both primers in the primer pair.
 4. A method according toclaim 1, wherein the reaction mixture further comprises a secondpolynucleotide having a modified nucleotide and wherein the singlestrand region is a single-stranded extension of less than 10 nucleotidessuch that the single stranded extension on the first polynucleotide iscomplementary to a second single-stranded extension on the secondpolynucleotide molecule.
 5. A method for joining a plurality of linearpolynucleotide molecules to form a single molecule, comprising: (a)forming a single-stranded extension on one or both ends of each of theplurality of polynucleotide molecules using the method of claim 1, suchthat at least one single-stranded extension on one polynucleotidemolecule is complementary to a single-stranded extension on anotherpolynucleotide molecule; and (b) allowing the plurality ofpolynucleotide molecules to associate to form the single molecule.
 6. Amethod for inserting a target molecule into a recipient molecule,comprising: (a) forming a first and a second single-stranded extensionhaving less than 10 nucleotides on a first and a second end of arecipient molecule using the method of claim 1, wherein the first andsecond single-stranded extension may be the same or different from eachother; (b) forming single-stranded extensions on the ends of a targetmolecule using the method of claim 1, wherein the single-strandedextensions are complementary to the first and the second single-strandedextension on the recipient molecule; and (c) allowing the recipientmolecule and the target molecule to associate to form a single molecule.7. A method according to claim 6, wherein the target molecule is aproduct of joining a plurality of polynucleotide molecules each having asingle strand extension generated according to claim
 1. 8. A methodaccording to claim 7, wherein the plurality of polynucleotides moleculescomprise A domains.
 9. A method according to claim 8, wherein the DNAdomains are exons.
 10. A method according to claim 1, wherein the singlestrand region is a single strand extension on one end of thepolynucleotide.